Methods and compositions involving mirna and mirna inhibitor molecules

ABSTRACT

The present invention concerns methods and compositions for introducing miRNA activity or function into cells using synthetic nucleic acid molecules. Moreover, the present invention concerns methods and compositions for identifying miRNAs with specific cellular functions that are relevant to therapeutic, diagnostic, and prognostic applications wherein synthetic miRNAs and/or miRNA inhibitors are used in library screening assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 60/683,736, filed on May 23, 2005, U.S.Provisional Patent Application Ser. No. 60/649,634, filed on Feb. 3,2005, and U.S. Provisional Patent Application Ser. No. 60/627,171, filedon Nov. 12, 2004 all of which are hereby incorporated by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING SUBMITTED ON A COMPACTDISC

The Sequence Listing is submitted on one compact disc (Copy 1), togetherwith a duplicate thereof (Copy 2), each created on Nov. 14, 2005, andeach containing one 173 kb file entitled “AMBI105US.txt.” The materialcontained on the compact disc is specifically incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of molecularbiology. More particularly, it concerns methods and compositionsinvolving nucleic acid molecules that simulate microRNA (miRNAs) andthat inhibit miRNAs. Methods and compositions involving synthetic miRNAsand miRNA inhibitor molecules are described. In addition, methods andcompositions for identifying miRNAs that contribute to cellularprocesses are also described. In addition, the identification of miRNAsthat contribute to cellular processes provides targets for therapeuticintervention as well as diagnostic and/or prognostic analysis.

2. Description of the Related Art

In 2001, several groups used a novel cloning method to isolate andidentify a large group of “microRNAs” (miRNAs) from C. elegans,Drosophila, and humans (Lagos-Quintana et al., 2001; Lau et al., 2001;Lee and Ambros, 2001). Several hundreds of miRNAs have been identifiedin plants and animals—including humans—which do not appear to haveendogenous siRNAs. Thus, while similar to siRNAs, miRNAs are nonethelessdistinct.

miRNAs thus far observed have been approximately 21-22 nucleotides inlength and they arise from longer precursors, which are transcribed fromnon-protein-encoding genes. See review of Carrington et al. (2003). Theprecursors form structures that fold back on each other inself-complementary regions; they are then processed by the nucleaseDicer in animals or DCL1 in plants. miRNA molecules interrupttranslation through precise or imprecise base-pairing with theirtargets.

miRNAs seem to be involved in gene regulation. Some miRNAs, includinglin-4 and let-7, inhibit protein synthesis by binding to partiallycomplementary 3′ untranslated regions (3′ UTRs) of target mRNAs. Others,including the Scarecrow miRNA found in plants, function like siRNA andbind to perfectly complementary mRNA sequences to destroy the targettranscript (Grishok et al., 2001).

Research on microRNAs is increasing as scientists are beginning toappreciate the broad role that these molecules play in the regulation ofeukaryotic gene expression. The two best understood miRNAs, lin-4 andlet-7, regulate developmental timing in C. elegans by regulating thetranslation of a family of key mRNAs (reviewed in Pasquinelli, 2002).Several hundred miRNAs have been identified in C. elegans, Drosophila,mouse, and humans. As would be expected for molecules that regulate geneexpression, miRNA levels have been shown to vary between tissues anddevelopmental states. In addition, one study shows a strong correlationbetween reduced expression of two miRNAs and chronic lymphocyticleukemia, providing a possible link between miRNAs and cancer (Calin,2002). Although the field is still young, there is speculation thatmiRNAs could be as important as transcription factors in regulating geneexpression in higher eukaryotes.

There are a few examples of miRNAs that play critical roles in celldifferentiation, early development, and cellular processes likeapoptosis and fat metabolism. lin-4 and let-7 both regulate passage fromone larval state to another during C. elegans development (Ambros,2003). mir-14 and bantam are drosophila miRNAs that regulate cell death,apparently by regulating the expression of genes involved in apoptosis(Brennecke et al., 2003, Xu et al., 2003). MiR14 has also beenimplicated in fat metabolism (Xu et al., 2003). Lsy-6 and miR-273 are C.elegans miRNAs that regulate asymmetry in chemosensory neurons (Chang etal., 2004). Another animal miRNA that regulates cell differentiation ismiR-181, which guides hematopoietic cell differentiation (Chen et al.,2004). These molecules represent the full range of animal miRNAs withknown functions. Enhanced understanding of the functions of miRNAs willundoubtedly reveal regulatory networks that contribute to normaldevelopment, differentiation, inter- and intra-cellular communication,cell cycle, angiogenesis, apoptosis, and many other cellular processes.Given their important roles in many biological functions, it is likelythat miRNAs will offer important points for therapeutic intervention ordiagnostic analysis.

Characterizing the functions of biomolecules like miRNAs often involvesintroducing the molecules into cells or removing the molecules fromcells and measuring the result. If introducing a miRNA into cellsresults in apoptosis, then the miRNA undoubtedly participates in anapoptotic pathway. Methods for introducing and removing miRNAs fromcells have been described. Two recent publications describe antisensemolecules that can be used to inhibit the activity of specific miRNAs(Meister et al., 2004; Hutvagner et al., 2004). Another publicationdescribes the use of plasmids that are transcribed by endogenous RNApolymerases and yield specific miRNAs when transfected into cells (Zenget al., 2002). These two reagent sets have been used to evaluate singlemiRNAs.

A limitation of the plasmid-based miRNA expression system is that thetransfection efficiencies for plasmids tend to be very low, with onlyapproximately 50% of cells expressing RNA from the plasmid in cells thatare easy to transfect. Transfection efficiencies for plasmids in primarycells are much lower, with fewer than 10% of cells typically expressingthe desired RNA. Therefore, there is a need for alternative compositionsand methods for introducing miRNA molecules into cells so that they canbe characterized and studied.

SUMMARY OF THE INVENTION

The present invention is based on the inventors' studies regarding theintroduction into cells of one or more nucleic acids that function likemiRNA or inhibit the activities of one or more miRNAs in cells tocharacterize their roles in various biological processes. The inventionconcerns nucleic acids that perform the activities of endogenous miRNAswhen introduced into cells. These nucleic acids are synthetic miRNA insome embodiments. The invention further concerns a library of syntheticmiRNAs specific to a variety of known miRNAs that can be used tointroduce sequentially or in combination one or more miRNAs into cellsin vitro or in vivo for the purpose of identifying miRNAs thatparticipate in cellular processes. The invention further involves alibrary of sequence-specific miRNA inhibitors that can be used toinhibit sequentially or in combination the activities of one or moremiRNAs in cells. The two libraries of miRNA-specific reagents are usedto introduce or eliminate specific miRNAs or combinations of miRNAs todefine the roles of miRNAs in cells.

The term “miRNA” is used according to its ordinary and plain meaning andrefers to a microRNA molecule found in eukaryotes that is involved inRNA-based gene regulation. See, e.g., Carrington et al., 2003, which ishereby incorporated by reference. The term will be used to refer to thesingle-stranded RNA molecule processed from a precursor. IndividualmiRNAs have been identified and sequenced in different organisms, andthey have been given names. Names of miRNAs and their sequences areprovided herein. Additionally, other miRNAs are known to those of skillin the art and can be readily implemented in embodiments of theinvention. The methods and compositions should not be limited to miRNAsidentified in the application, as they are provided as examples, notnecessarily as limitations of the invention.

The present invention concerns, in some embodiments of the invention,short nucleic acid molecules that function as miRNAs or as inhibitors ofmiRNA in a cell. The term “short” refers to a length of a singlepolynucleotide that is 150 nucleotides or fewer. The nucleic acidmolecules are synthetic. The term “synthetic” means the nucleic acidmolecule is isolated and not identical in sequence (the entire sequence)and/or chemical structure to a naturally-occurring nucleic acidmolecule, such as an endogenous precursor miRNA molecule. While in someembodiments, nucleic acids of the invention do not have an entiresequence that is identical to a sequence of a naturally-occurringnucleic acid, such molecules may encompass all or part of anaturally-occurring sequence. It is contemplated, however, that asynthetic nucleic acid administered to a cell may subsequently bemodified or altered in the cell such that its structure or sequence isthe same as non-synthetic or naturally occurring nucleic acid, such as amature miRNA sequence. For example, a synthetic nucleic acid may have asequence that differs from the sequence of a precursor miRNA, but thatsequence may be altered once in a cell to be the same as an endogenous,processed miRNA. The term “isolated” means that the nucleic acidmolecules of the invention are initially separated from different (interms of sequence or structure) and unwanted nucleic acid molecules suchthat a population of isolated nucleic acids is at least about 90%homogenous, and may be at least about 95, 96, 97, 98, 99, or 100%homogenous with respect to other polynucleotide molecules. In manyembodiments of the invention, a nucleic acid is isolated by virtue of ithaving been synthesized in vitro separate from endogenous nucleic acidsin a cell. It will be understood, however, that isolated nucleic acidsmay be subsequently mixed or pooled together.

Of course, it is understood that a “synthetic nucleic acid” of theinvention means that the nucleic acid does not have a chemical structureor sequence of a naturally occurring nucleic acid. Consequently, it willbe understood that the term “synthetic miRNA” refers to a “syntheticnucleic acid” that functions in a cell or under physiological conditionsas a naturally occurring miRNA.

While many of the embodiments of the invention involve synthetic miRNAsor synthetic nucleic acids, in some embodiments of the invention, thenucleic acid molecule(s) need not be “synthetic.” In certainembodiments, a non-synthetic miRNA employed in methods and compositionsof the invention may have the entire sequence and structure of anaturally occurring miRNA precursor or the mature miRNA. For example,non-synthetic miRNAs used in methods and compositions of the inventionmay not have one or more modified nucleotides or nucleotide analogs. Inthese embodiments, the non-synthetic miRNA may or may not berecombinantly produced. In particular embodiments, the nucleic acid inmethods and/or compositions of the invention is specifically a syntheticmiRNA and not a non-synthetic miRNA (that is, not an miRNA thatqualifies as “synthetic”); though in other embodiments, the inventionspecifically involves a non-synthetic miRNA and not a synthetic miRNA.Any embodiments discussed with respect to the use of synthetic miRNAscan be applied with respect to non-synthetic miRNAs, and vice versa.

It will be understood that the term “naturally occurring” refers tosomething found in an organism without any intervention by a person; itcould refer to a naturally-occurring wildtype or mutant molecule. Insome embodiments a synthetic miRNA molecule does not have the sequenceof a naturally occurring miRNA molecule. In other embodiments, asynthetic miRNA molecule may have the sequence of a naturally occurringmiRNA molecule, but the chemical structure of the molecule, particularlyin the part unrelated specifically to the precise sequence (non-sequencechemical structure) differs from chemical structure of the naturallyoccurring miRNA molecule with that sequence. In some cases, thesynthetic miRNA has both a sequence and non-sequence chemical structurethat are not found in a naturally-occurring miRNA. Moreover, thesequence of the synthetic molecules will identify which miRNA iseffectively being provided or inhibited; the endogenous miRNA will bereferred to as the “corresponding miRNA.” Corresponding miRNA sequencesthat can be used in the context of the invention include, but are notlimited to, those sequences in SEQ ID NOs: 1-593 and those miRNAs listedin the appendix. In addition synthetic nucleic acids of the inventionmay include SEQ ID NOs:594-703 as well as any other miRNA sequence,miRNA precursor sequence, or any sequence complementary thereof. In someembodiments, the sequence is or is derived from a probe sequenceidentified in the appendix to target the particular miRNA (or set ofmiRNAs) that can be used with that probe sequence.

Synthetic miRNA of the invention are RNA or RNA analogs in someembodiments of the invention. mRNA inhibitors may be DNA or RNA, oranalogs thereof miRNA and miRNA inhibitors of the invention arecollectively referred to as “synthetic nucleic acids.”

In some embodiments, there is a synthetic miRNA having a length ofbetween 17 and 130 residues. The present invention concerns syntheticmiRNA molecules that are, are at least, or are at most 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, or 130 residues in length,or any range derivable therein.

In certain embodiments, synthetic miRNA have a) an “miRNA region” whosesequence from 5′ to 3′ is identical to a mature miRNA sequence, and b) a“complementary region” whose sequence from 5′ to 3′ is between 60% and100% complementary to the miRNA sequence. In certain embodiments, thesesynthetic miRNA are also isolated, as defined above. The term “miRNAregion” refers to a region on the synthetic miRNA that is at least 90%identical to the entire sequence of a mature, naturally occurring miRNAsequence. In certain embodiments, the miRNA region is or is at least 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6,99.7, 99.8, 99.9 or 100% identical to the sequence of anaturally-occurring miRNA.

The term “complementary region” refers to a region of a synthetic miRNAthat is or is at least 60% complementary to the mature, naturallyoccurring miRNA sequence that the miRNA region is identical to. Thecomplementary region is or is at least 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2,99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or anyrange derivable therein. With single polynucleotide sequences, there isa hairpin loop structure as a result of chemical bonding between themiRNA region and the complementary region. In other embodiments, thecomplementary region is on a different nucleic acid molecule than themiRNA region, in which case the complementary region is on thecomplementary strand and the miRNA region is on the active strand.

In other embodiments of the invention, there are synthetic nucleic acidsthat are miRNA inhibitors. An miRNA inhibitor is between about 17 to 25nucleotides in length and comprises a 5′ to 3′ sequence that is at least90% complementary to the 5′ to 3′ sequence of a mature miRNA. In certainembodiments, an miRNA inhibitor molecule is 17, 18, 19, 20, 21, 22, 23,24, or 25 nucleotides in length, or any range derivable therein.Moreover, an miRNA inhibitor has a sequence (from 5′ to 3′) that is oris at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3,99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any rangederivable therein, to the 5′ to 3′ sequence of a mature miRNA,particularly a mature, naturally occurring miRNA. Probe sequences formiRNAs are disclosed in the appendix. While they have more sequence thanan miRNA inhibitor, one of skill in the art could use that portion ofthe probe sequence that is complementary to the sequence of a maturemiRNA as the sequence for an miRNA inhibitor. Table 1 indicates what themature sequence of an miRNA is. Moreover, that portion of the probesequence can be altered so that it is still 90% complementary to thesequence of a mature miRNA.

In some embodiments, of the invention, a synthetic miRNA contains one ormore design elements. These design elements include, but are not limitedto: i) a replacement group for the phosphate or hydroxyl of thenucleotide at the 5′ terminus of the complementary region; ii) one ormore sugar modifications in the first or last 1 to 6 residues of thecomplementary region; or, iii) noncomplementarity between one or morenucleotides in the last 1 to 5 residues at the 3′ end of thecomplementary region and the corresponding nucleotides of the miRNAregion.

In certain embodiments, a synthetic miRNA has a nucleotide at its 5′ endof the complementary region in which the phosphate and/or hydroxyl grouphas been replaced with another chemical group (referred to as the“replacement design”). In some cases, the phosphate group is replaced,while in others, the hydroxyl group has been replaced. In particularembodiments, the replacement group is biotin, an amine group, a loweralkylamine group, an acetyl group, 2′O-Me (2′oxygen-methyl), DMTO(4,4′-dimethoxytrityl with oxygen), fluoroscein, a thiol, or acridine,though other replacement groups are well known to those of skill in theart and can be used as well. This design element can also be used withan miRNA inhibitor.

Additional embodiments concern a synthetic miRNA having one or moresugar modifications in the first or last 1 to 6 residues of thecomplementary region (referred to as the “sugar replacement design”). Incertain cases, there is one or more sugar modifications in the first 1,2, 3, 4, 5, 6 or more residues of the complementary region, or any rangederivable therein. In additional cases, there is one or more sugarmodifications in the last 1, 2, 3, 4, 5, 6 or more residues of thecomplementary region, or any range derivable therein, have a sugarmodification. It will be understood that the terms “first” and “last”are with respect to the order of residues from the 5′ end to the 3′ endof the region. In particular embodiments, the sugar modification is a2′O-Me modification. In further embodiments, there is one or more sugarmodifications in the first or last 2 to 4 residues of the complementaryregion or the first or last 4 to 6 residues of the complementary region.This design element can also be used with an miRNA inhibitor. Thus, anmiRNA inhibitor can have this design element and/or a replacement groupon the nucleotide at the 5′ terminus, as discussed above.

In other embodiments of the invention, there is a synthetic miRNA inwhich one or more nucleotides in the last 1 to 5 residues at the 3′ endof the complementary region are not complementary to the correspondingnucleotides of the miRNA region (“noncomplementarity”) (referred to asthe “noncomplementarity design”). The noncomplementarity may be in thelast 1, 2, 3, 4, and/or 5 residues of the complementary miRNA. Incertain embodiments, there is noncomplementarity with at least 2nucleotides in the complementary region.

It is contemplated that synthetic miRNA of the invention have one ormore of the replacement, sugar modification, or noncomplementaritydesigns. In certain cases, synthetic RNA molecules have two of them,while in others these molecules have all three designs in place.

The miRNA region and the complementary region may be on the same orseparate polynucleotides. In cases in which they are contained on or inthe same polynucleotide, the miRNA molecule will be considered a singlepolynucleotide. In embodiments in which the different regions are onseparate polynucleotides, the synthetic miRNA will be considered to becomprised of two polynucleotides.

When the RNA molecule is a single polynucleotide, there is a linkerregion between the miRNA region and the complementary region. In someembodiments, the single polynucleotide is capable of forming a hairpinloop structure as a result of bonding between the miRNA region and thecomplementary region. The linker constitutes the hairpin loop. It iscontemplated that in some embodiments, the linker region is, is atleast, or is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40 residues in length, or any range derivabletherein. In certain embodiments, the linker is between 3 and 30 residues(inclusive) in length.

In addition to having an miRNA region and a complementary region, theremay be flanking sequences as well at either the 5′ or 3′ end of theregion. In some embodiments, there is or is at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10 nucleotides or more, or any range derivable therein,flanking one or both sides of these regions.

The present invention also concerns a collection of synthetic nucleicacid molecules, referred to as a library. A collection may contain,contain at least or contain at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521,522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535,536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549,550 or more different types (by structure and/or sequence) of nucleicacids. Libraries may contain synthetic miRNAs and/or miRNA inhibitors.

Embodiments involving libraries and methods of using nucleic acids ofthe invention may be applied to miRNA and miRNA inhibitors. Thus, anyembodiment discussed with respect to nucleic acids of the invention maygenerally be applicable to miRNA and miRNA inhibitor molecules, and viceversa. Moreover, embodiments discussed with respect to miRNA may beapplied to miRNA inhibitors and vice versa.

The present invention also concerns methods of characterizing an miRNAactivity or function in a cell. In some embodiments, a method comprises:a) introducing into one or more cells a synthetic miRNA molecule; and b)comparing one or more characteristics of cell(s) having the RNA moleculewith cells in which the synthetic miRNA molecule has not beenintroduced. In certain embodiments, the cells with the synthetic miRNAmay be compared to cells in which a different molecule was introduced(such as a negative control that does not include an miRNA region or hasan miRNA region for a different miRNA). It is contemplated that thecompared cells need not be evaluated at the same time. In fact, thecomparison cells need not have been cultured at the same time; one mayrefer to a report or previous observation.

Other methods include reducing or eliminating activity of one or moremiRNAs from a cell comprising: a) introducing into a cell an miRNAinhibitor. In certain embodiment, methods also include comparing one ormore characteristics of a cell having the miRNA inhibitor with a cellnot having the miRNA inhibitor.

The synthetic nucleic acids discussed above and herein can be used inmethods of the invention. Thus, in certain embodiments, the methodsinvolve synthetic nucleic acids with the different designs in them.

Characteristics of cells that may be evaluated are not limited. Theyinclude the following characteristics and characteristics associatedwith the following: cell proliferation, mitotic index, cell cycle,apoptosis, motility, adhesion, signal transduction, proteinlocalization, gene expression, RNA localization, cell division, DNAreplication, post-translational modification, differentiation,de-differentiation, transcriptional activation, protein activation,angiogenesis, metabolism (energy production and/or consumption), proteindegradation, chromatin condensation, microtubule production, DNAreplication, recombination, and DNA repair functions. It is contemplatedthat these characteristics may be relevant globally to the cell (forexample, overall protein production reduced) or to individual species inthe cell (for example, induction of a specific protein(s)).

It is contemplated that this method may be applied with respect to avariety of different synthetic and/or nonsynthetic miRNAs in separate orthe same cells. In some cases, the following numbers of differentsynthetic miRNA molecules may be introduced into different cells: 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 297, 298, 299, 300 or more, or any rangederivable therein (or at least or at most these numbers). The inventionis not limited by cell type. It is contemplated that any cell expressingmiRNA or any cell having a characteristic altered by an miRNA isamenable to the methods and compositions of the invention. Use of two ormore miRNAs may be combined in a single pharmaceutical composition as acocktail or may be used in any therapeutic, diagnostic or prognosticmethod of the invention. It is contemplated that methods of theinvention may involve, involve at least, or involve at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278,279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,293, 294, 295, 296, 297, 298, 299, 300 or more, or any range derivabletherein, nucleic acid molecules corresponding to different miRNAs. Suchnucleic acid molecules include synthetic miRNAs molecules, nonsyntheticmiRNA molecules, and miRNA inhibitors.

In some embodiments, it may be useful to know whether a cell expresses aparticular miRNA endogenously or whether such expression is affectedunder particular conditions or when it is in a particular disease state.Thus, in some embodiments of the invention, methods include assaying thecell for the presence of the miRNA that is effectively being introducedby the synthetic miRNA molecule or inhibited by an miRNA inhibitor.Consequently, in some embodiments, methods include a step of generatingan miRNA profile for a sample. The term “miRNA profile” refers to a setof data regarding the expression pattern for a plurality of miRNAs inthe sample; it is contemplated that the miRNA profile can be obtainedusing an miRNA array. In some embodiments of the invention, an miRNAprofile is generated by steps that include: a) labeling miRNA in thesample; b) hybridizing the miRNA to an miRNA array; and, c) determiningmiRNA hybridization to the array, wherein an miRNA profile is generated.See U.S. Provisional Patent Application 60/575,743 and the U.S.Provisional Patent Application 60/649,584, and U.S. patent applicationSer. No. 11/141,707, all of which are hereby incorporated by reference.

Additionally, a cell that is introduced with a synthetic miRNA or anmiRNA inhibitor may be subsequently evaluated or assayed for the amountof endogenous or exogenous miRNA or miRNA inhibitor. Any cell type iscontemplated for use with the invention. The cell may be from or in amammal, such as a monkey, horse, cow, pig, sheep, dog, cat, rabbit,mouse, rat, or human.

In other methods of the invention, a step of synthesizing or obtainingthe synthetic RNA molecule is included.

In additional embodiments, the synthetic nucleic acid is introduced intothe cell by calcium phosphate transfection, lipid transfection,electroporation, microinjection, or injection. In addition, a cell maybe in a subject, which may be a patient or an animal model. In thiscase, synthetic nucleic acids can be administered to the subject orpatient using modes of administration that are well known to those ofskill in the art, particularly for therapeutic applications. It isparticularly contemplated that a patient is human or any other mammal oranimal having miRNA.

The present invention also concerns inducing certain cellularcharacteristics by providing to a cell a particular nucleic acid, suchas a specific synthetic miRNA molecule or a synthetic miRNA inhibitormolecule. However, in methods of the invention, the miRNA molecule ormiRNA inhibitor need not be synthetic. They may have a sequence that isidentical to a naturally occurring miRNA or they may not have any designmodifications. In certain embodiments, the miRNA molecule and/or anmiRNA inhibitor are synthetic, as discussed above.

The particular nucleic acid molecule provided to the cell is understoodto correspond to a particular miRNA in the cell, and thus, the miRNA inthe cell is referred to as the “corresponding miRNA.” In situations inwhich a named miRNA molecule is introduced into a cell, thecorresponding miRNA will be understood to be the induced miRNA. It iscontemplated, however, that the miRNA molecule provided introduced intoa cell is not a mature miRNA but is capable of becoming a mature miRNAunder the appropriate physiological conditions. In cases in which aparticular corresponding miRNA is being inhibited by a miRNA inhibitor,the particular miRNA will be referred to as the targeted miRNA. It iscontemplated that multiple corresponding miRNAs may be involved. Inparticular embodiments, more than one miRNA molecule is introduced intoa cell. Moreover, in other embodiments, more than one miRNA inhibitor isintroduced into a cell. Furthermore, a combination of miRNA molecule(s)and miRNA inhibitor(s) may be introduced into a cell.

Methods include identifying a cell or patient in need of inducing thosecellular characteristics. Also, it will be understood that an amount ofa synthetic nucleic acid that is provided to a cell or organism is an“effective amount,” which refers to an amount needed to achieve adesired goal, such as inducing a particular cellular characteristic(s).

In certain embodiments of the methods include providing or introducingto a cell a nucleic acid molecule corresponding to a mature miRNA in thecell in an amount effective to achieve a desired physiological result.Such methods are disclosed herein. Moreover, methods of the inventioninvolve diagnosing a patient based on an miRNA expression profile. Incertain embodiments, the elevation or reduction in the level ofexpression of a particular miRNA in a cell is correlated with a diseasestate compared to the expression level of that miRNA in a normal cell.This correlation allows for diagnostic methods to be carried out whenthat the expression level of an miRNA is measured in a biological samplebeing assessed and then compared to the expression level of a normalcell.

In these different methods, the corresponding miRNA involved in themethod may be one or more of at least the following: Let 7a, let 7a-1,let 7b, let 7b-1, let-7c, let-7d, let 7g, miR-1, miR-1-d, miR-1-2,miR-9, miR-10a, miR-10b, miR-15a, miR-16, miR-17, miR-17-3p, miR-18,miR-19a, miR-20, miR-21, miR-22, miR-23, miR-23a, miR-23b, miR-24,miR-25, miR-26a, miR-27a, miR-28, miR-29a, miR-29b, miR-30a-3p, miR-30a,miR-30e-5p, miR-31, miR-32, miR-34a, miR-92, miR-93, miR-95, miR-96,miR-98, miR-99a, miR-100, miR-101, miR-105, miR-106, miR-107, miR-108,miR-122, miR-124, miR-125, miR-125b, miR-126, miR-127, miR-128, miR-129,miR-130, miR-130a, miR-133, miR-133a, miR-133a-2, miR-133b, miR-134,miR-135, miR-137, miR-138, miR-139, miR-140, miR-141, miR-142, miR-143,miR-145, miR-147, miR-148, miR-149, miR-150, miR-152, miR-153, miR-154,miR-155, miR-181, miR-182, miR-183, miR-184, miR-186, miR-187, miR-188,miR-190, miR-191, miR-192, miR-193, miR-194, miR-195, miR-196, miR-197,miR-198, miR-199, miR-199a-1, miR-200b, miR-201, miR-203, miR-204,miR-206, miR-207, miR-208, miR-210, miR-211, miR-212, miR-213, miR-214,miR-215, miR-216, miR-217, miR-218, miR-222, miR-223, miR-224,miR-291-3p, miR-292, miR-292-3p, miR-293, miR-294, miR-295, miR-296,miR-297, miR-298, miR-299, miR-320, miR-321, miR-322, miR-324, miR-325,miR-326, miR-328, miR-329, miR-330, miR-331, miR-333, miR-335, miR-337,miR-338, miR-340, miR-341, miR-342, miR-344, miR-345, miR-346, miR-350,miR-367, miR-368, miR-369, miR-370, miR-371, miR-373, miR-380-3p,miR-409, miR-410, or miR-412.

Moreover, methods can involve providing synthetic or nonsynthetic miRNAmolecules. It is contemplated that in these embodiments, methods may ormay not be limited to providing only one or more synthetic miRNAmolecules or only on or more nonsynthetic miRNA molecules. Thus, incertain embodiments, methods may involve providing both synthetic andnonsynthetic miRNA molecules. In this situation, a cell or cells aremost likely provided a synthetic miRNA molecule corresponding to aparticular miRNA and a nonsynthetic miRNA molecule corresponding to adifferent miRNA. Furthermore, any method articulated a list of miRNAsusing Markush group language may be articulated without the Markushgroup language and a disjunctive article (i.e., or) instead, and viceversa.

In some embodiments, there is a method for reducing or inhibiting cellproliferation in a cell comprising introducing into or providing to thecell an effective amount of i) an miRNA inhibitor molecule or ii) asynthetic or nonsynthetic miRNA molecule that corresponds to an miRNAsequence. In certain embodiments the methods involves introducing intothe cell an effective amount of i) an miRNA inhibitor molecule having a5′ to 3′ sequence that is at least 90% complementary to the 5′ to 3′sequence of a mature miRNA selected from the group consisting of:mir-31, mir-92, mir-99a, mir-100, mir-125a, mir-129, mir-130a, mir-150,mir-187, miR-190, miR-191, miR-193, miR 204, mir-210, mir-211, mir-212,mir-213, mir-215, mir-216, mir-217, miR 218, mir-224, mir-292, mir-294,mir-320, mir-324, mir-325, mir-326, mir-330, mir-331, mir-338, mir-341,mir-369, and mir-370; or ii) a synthetic or nonsynthetic miRNA moleculethat corresponds to an miRNA sequence selected from the group consistingof: miR-15a, miR-16, miR 21, miR 24, miR-96, miR-101, miR-105, miR-124,miR-126, miR-142, miR-147, miR-192, miR-194, miR-206, miR-215, ormiR-346.

In addition or alternatively, any of the following may be included inthe group from which the miRNA inhibitor molecule (i) may be chosen:Let-7a, Let-7b, Let-7c, Let-7d, Let-7g, miR-7, mir-9, miR-10a, miR-10b,miR-18, miR-19a, miR-17-3p, miR-20, miR-23b, mir-25, miR-26a, miR-26a,mir-30e-5p, mir-31, mir-32, mir-92, mir-93, miR-100, miR-125a, miR-125b,mir-127, miR-128, miR-129, mir-130a, mir-135, mir-138, mir-139, miR-140,mir-141, mir-143, mir-145, mir-146, miR-150, mir-154, mir-155, mir-181a,miR-182, mir-186, miR-187, miR-188, mir-190, mir-191, mir-193, mir-196,mir-197, mir-198, mir-199, mir-201, mir-204, mir-216, mir-218, miR-223,mir-293, miR-291-3p, miR-294, miR-295, miR-322, mir-333, mir-335,mir-338, mir-341, mir-350, mir-369, miR-373, mir-410, and mir-412. Inaddition or alternatively, any of the following may be included in thegroup from which the miRNA molecule that corresponds to an miRNAsequence (ii) may be chosen: let7a-1, Let-7a, Let-7b, let7b-1, let7c,let7d, Let-7g, mir-9, mir-10a, mir-10b, mir-15a, mir-16, mir-21,mir-23a, mir-23b, mir-24, mir-25, mir-92, mir-95, mir-133a, mir-133a-2,mir-133b, mir-142, mir-152, mir-153, mir-155, mir-181a, mir-182,mir-183, mir-184, mir-186, mir-187, mir-191, mir-193, mir-194, mir-196,mir-199a-1, mir-200b, mir-204, mir-206, mir-211, mir-222, mir-223,mir-298, mir-328, mir-342, mir-371, and mir-412.

In other words, methods involve providing a synthetic miRNA inhibitorhaving a sequence that is at least 90% complementary to the 5′ to 3′sequence of a corresponding miRNA that is mir-31, mir-92, mir-99a,mir-100, mir-125a, mir-129, mir-130a, mir-150, mir-187, miR-190,miR-191, miR-193, miR 204, mir-210, mir-211, mir-212, mir-213, mir-215,mir-216, mir-217, miR 218, mir-224, mir-292, mir-294, mir-320, mir-324,mir-325, mir-326, mir-330, mir-331, mir-338, mir-341, mir-369, mir-370,Let-7a, Let-7b, Let-7c, Let-7d, Let-7g, miR-7, mir-9, miR-10a, miR-10b,miR-18, miR-19a, miR-17-3p, miR-20, miR-23b, mir-25, miR-26a, miR-26a,mir-30e-5p, mir-31, mir-32, mir-92, mir-93, miR-100, miR-125a, miR-125b,mir-127, miR-128, miR-129, mir-130a, mir-135, mir-138, mir-139, miR-140,mir-141, mir-143, mir-145, mir-146, miR-150, mir-154, mir-155, mir-181a,miR-182, mir-186, miR-187, miR-188, mir-190, mir-191, mir-193, mir-196,mir-197, mir-198, mir-199, mir-201, mir-204, mir-216, mir-218, miR-223,mir-293, miR-291-3p, miR-294, miR-295, miR-322, mir-333, mir-335,mir-338, mir-341, mir-350, mir-369, miR-373, mir-410, or mir-412.Alternatively or additionally, methods involve providing a synthetic ornonsynthetic miRNA molecule that corresponds to miR-15a, miR-16, miR 21,miR 24, miR-96, miR-01, miR-105, miR-124, miR-126, miR-142, miR-147,miR-192, miR-194, miR-206, miR-215, miR-346, let7a-1, Let-7a, Let-7b,let7b-1, let7c, let7d, Let-7g, mir-9, mir-10a, mir-10b, mir-15a, mir-16,mir-21, mir-23a, mir-23b, mir-24, mir-25, mir-92, mir-95, mir-133a,mir-133a-2, mir-133b, mir-142, mir-152, mir-153, mir-155, mir-181a,mir-182, mir-183, mir-184, mir-186, mir-187, mir-191, mir-193, mir-194,mir-196, mir-199a-1, mir-200b, mir-204, mir-206, mir-211, mir-222,mir-223, mir-298, mir-328, mir-342, mir-371, or mir-412. Methods forreducing or inhibiting cell proliferation can be used as a treatment fordiseases and conditions that include, but are not limited to,hyperproliferative diseases, such as cancer.

The present invention also concerns methods for inducing or increasingcell proliferation in a cell comprising introducing into or providing tothe cell an effective amount of i) an miRNA inhibitor molecule or ii) asynthetic or nonsynthetic miRNA molecule that corresponds to an miRNAsequence. In certain embodiments the methods involves introducing intoor providing the cell an effective amount of i) an miRNA inhibitorcorresponding to let7a-1, Let-7a, Let-7b, let7b-1, let7c, let7d, Let-7g,mir-9, mir-10a, mir-10b, mir-15a, mir-16, mir-21, mir-23a, mir-23b,mir-24, mir-25, mir-92, mir-95, mir-133a, mir-133a-2, mir-133b, mir-142,mir-152, mir-153, mir-155, mir-181a, mir-182, mir-183, mir-184, mir-186,mir-187, mir-191, mir-193, mir-194, mir-196, mir-199a-1, mir-200b,mir-204, mir-206, mir-211, mir-222, mir-223, mir-298, mir-328, mir-342,mir-371, and mir-412; or ii) a miRNA molecule corresponding to Let-7a,Let-7b, Let-7c, Let-7d, Let-7g, miR-7, mir-9, miR-10a, miR-10b, miR-15a,miR-18, miR-19a, miR-17-3p, miR-20, miR-23b, mir-25, miR-26a, miR-26a,mir-30e-5p, mir-31, mir-32, mir-92, mir-93, miR-100, miR-125a, miR-125b,miR-126, mir-127, miR-128, miR-129, mir-130a, mir-135, mir-138, mir-139,miR-140, mir-141, mir-143, mir-145, mir-146, miR-150, mir-154, mir-155,mir-181a, miR-182, mir-186, miR-187, miR-188, mir-190, mir-191, mir-193,mir-194, mir-196, mir-197, mir-198, mir-199, mir-201, mir-204, mir-216,mir-218, miR-223, mir-293, miR-291-3p, miR-294, miR-295, miR-322,mir-333, mir-335, mir-338, mir-341, mir-350, mir-369, miR-373, mir-410,and mir-412. Alternatively or additionally, the group of miRNAinhibitors includes miR-15a, miR-16, miR 21, miR 24, miR-96, miR-101,miR-105, miR-124, miR-126, miR-142, miR-147, miR-192, miR-194, miR-206,miR-215, or miR-346 and the group of miRNAs molecules corresponding tomiRNAs includes mir-31, mir-92, mir-99a, mir-100, mir-125a, mir-129,mir-130a, mir-150, mir-187, miR-190, miR-191, miR-193, miR 204, mir-210,mir-211, mir-212, mir-213, mir-215, mir-216, mir-217, miR 218, mir-224,mir-292, mir-294, mir-320, mir-324, mir-325, mir-326, mir-330, mir-331,mir-338, mir-341, mir-369, and mir-370.

Such methods can be used for the treatment of wounds, burns, ischemia,or any other condition, disease, or symptom in which cell proliferationis desirable.

It will be understood in methods of the invention that a cell or otherbiological matter such as an organism (including patients) can beprovided an miRNA or miRNA molecule corresponding to a particular miRNAby administering to the cell or organism a nucleic acid molecule thatfunctions as the corresponding miRNA once inside the cell. The form ofthe molecule provided to the cell may not be the form that acts an miRNAonce inside the cell. Thus, it is contemplated that in some embodiments,biological matter is provided a synthetic miRNA or a nonsynthetic miRNA,such as one that becomes processed into a mature and active miRNA onceit has access to the cell's miRNA processing machinery. In certainembodiments, it is specifically contemplated that the miRNA moleculeprovided to the biological matter is not a mature miRNA molecule but anucleic acid molecule that can be processed into the mature miRNA onceit is accessible to miRNA processing machinery. The term “nonsynthetic”in the context of miRNA means that the miRNA is not “synthetic,” asdefined herein. Furthermore, it is contemplated that in embodiments ofthe invention that concern the use of synthetic miRNAs, the use ofcorresponding nonsynthetic miRNAs is also considered an aspect of theinvention, and vice versa.

In other embodiments, the methods involve reducing cell viabilitycomprising introducing into or providing to the cell an effective amountof i) an miRNA inhibitor molecule or ii) a synthetic or nonsyntheticmiRNA molecule that corresponds to an miRNA sequence. In certainembodiments the methods involves introducing into one or more cells aneffective amount of i) an miRNA inhibitor corresponding to miR-107,miR-133, miR-137, miR-152, miR-155, miR-181a, miR-191, miR-203, ormiR-215; or ii) an miRNA molecule corresponding to let-7a, let-7b,mir-1, mir-7, miR-10b, miR-17-3p, miR-19a, mir-23, mir-24, mir-27a,miR-29a, miR-30a-3p, mir-31, mir-32, miR-34a, miR-101, miR-107, miR-108,miR-122, mir-124, miR-133a, miR-134, miR-135, miR-139, mir-140, miR-141,miR-145, mir-150, mir-192, mir-193, mir-195, mir-206, mir-208, mir-210,mir-210, mir-292-3p, mir-293, mir-297, mir-299, mir-329, mir-337,mir-337, mir-345, mir-346, and mir-409. Alternatively or additionally,the group of miRNA inhibitors (group i) includes let-7a, let-7b, let-7c,let-7d, let-7g, miR-10a, miR-10b, miR-15a, miR-17-3p, miR-18, miR-19a,miR-20, mir-23a, mir-23b, mir-24, miR-25, miR-26a, mir-32, miR-107,miR-125a, miR-126, mir-128, miR-129, miR-133, miR-137, mir-139, miR-143,miR-152, miR-155, miR-181a, miR-182, miR-191, miR-203, miR-215, andmir-331

Other aspects of the invention include a method for increasing cellviability comprising introducing into or providing to the cell aneffective amount of i) an miRNA inhibitor molecule or ii) a synthetic ornonsynthetic miRNA molecule that corresponds to an miRNA sequence. Incertain embodiments the methods involves introducing into one or morecells an effective amount of i) an miRNA inhibitor corresponding tomiR-7, miR-19a, miR-23, miR-24, miR-27a, miR-31, miR-32, miR-134,miR-140, miR-150, miR-192, or miR-193; or ii) an miRNA moleculecorresponding to let-7a, let-7b, let-7c, let-7d, let-7g, miR-10a,miR-10b, miR-15a, miR-17-3p, miR-18, miR-19a, miR-20, mir-23a, mir-23b,mir-24, miR-25, miR-26a, mir-32, miR-107, miR-125a, miR-126, mir-128,miR-129, miR-133, miR-137, mir-139, miR-143, miR-152, miR-155, miR-181a,miR-182, miR-191, miR-203, miR-215, and mir-331. Alternatively oradditionally, the group of miRNA inhibitors (group i) includes let-7a,let-7b, mir-1, mir-7, miR-10b, miR-17-3p, miR-19a, mir-23, mir-24,mir-27a, miR-29a, miR-30a-3p, mir-31, mir-32, miR-34a, miR-101, miR-107,miR-108, miR-122, mir-124, miR-133a, miR-134, miR-135, miR-139, mir-140,miR-141, miR-145, mir-150, mir-192, mir-193, mir-195, mir-206, mir-208,mir-210, mir-210, mir-292-3p, mir-293, mir-297, mir-299, mir-329,mir-337, mir-337, mir-345, mir-346, or mir-409, and the group of miRNAsmolecules corresponding to miRNAs (group ii) includes. The presentinvention also concerns a method for inducing apoptosis in a cellcomprising introducing into or providing to the cell an effective amountof i) an miRNA inhibitor molecule or ii) a synthetic or nonsyntheticmiRNA molecule that corresponds to an miRNA sequence. In certainembodiments the methods involves introducing into the cell an effectiveamount of i) an miRNA inhibitor corresponding to miR-31 or miR-214; orii) an miRNA molecule corresponding to let-7b, let-7g, mir-1, mir-1d,mir-7, mir-10a, miR-10b, miR-17-3p, miR-19a, miR-28, miR-28, miR-28,miR-29a, miR-32, miR-34a, miR-122, mir-148, mir-149, mir-154, mir-184,mir-186, mir-188, mir-192, mir-195, mir-196, mir-199a, mir-204, mir-208,mir-210, mir-211, mir-212, mir-214, mir-215, mir-216, mir-217, mir-218,mir-293, mir-296, mir-299, mir-321, mir-328, or mir-344. Alternativelyor additionally, the group of miRNA inhibitors (group i) includesLet-7b, mir-21, mir-23b, mir-25, miR-26a, mir-28, mir-29a, mir-31,miR-32, mir-30a-3p, mir-34a, mir-96, miR-98, mir-100, mir-101, mir-105,mir-108, miR-125b, miR-126, mir-126, mir-128, mir-137, miR-143, miR-155,mir-207, mir-214, mir-216, mir-223, mir-292-3p, mir-328, mir-335,mir-340, mir-341, mir-367, mir-368, mir-380-3p, and mir-410.

Methods for inducing apoptosis have a number of therapeutic applicationsincluding, but not limited to, the treatment of cancer.

Other embodiments of the invention involve a method for inhibitingapoptosis in a cell comprising introducing into or providing to the cellan effective amount of i) an miRNA inhibitor molecule or ii) a syntheticor nonsynthetic miRNA molecule that corresponds to an miRNA sequence. Incertain embodiments the methods involves introducing into the cell aneffective amount of i) an miRNA inhibitor corresponding to miR-7,miR-1-2, miR-148, miR-195, miR-196, miR-199a, miR-204, miR-210, miR-211,miR-212, miR-215, miR-216, miR-218, miR-296, or miR-321; or ii) an miRNAmolecule corresponding to Let-7b, mir-21, mir-23b, mir-25, miR-26a,mir-28, mir-29a, mir-31, miR-32, mir-30a-3p, mir-34a, mir-96, miR-98,mir-100, mir-101, mir-105, mir-108, miR-125b, miR-126, mir-126, mir-128,mir-137, miR-143, miR-155, mir-207, mir-214, mir-216, mir-223,mir-292-3p, mir-328, mir-335, mir-340, mir-341, mir-367, mir-368,mir-380-3p, or mir-410. Alternatively or additionally, the group ofmiRNA inhibitors (group i) includes let-7b, let-7g, mir-1, mir-1d,mir-7, mir-10a, miR-10b, miR-17-3p, miR-19a, miR-28, miR-28, miR-28,miR-29a, miR-32, miR-34a, miR-122, mir-148, mir-149, mir-154, mir-184,mir-186, mir-188, mir-192, mir-195, mir-196, mir-199a, mir-204, mir-208,mir-210, mir-211, mir-212, mir-214, mir-215, mir-216, mir-217, mir-218,mir-293, mir-296, mir-299, mir-321, mir-328, or mir-344.

The present invention also concerns using miRNA compositions to treatdiseases or conditions or to prepare therapeutics for the treatment ofdiseases or conditions. In some embodiments, the invention involves oneor more human miRNA selected from the group consisting of let-7,miR-10a, miR-15a, miR-16, miR-17, miR-21, miR-22, miR-23, miR-24,miR-26a, miR-29b, miR-30a, miR-96, miR-101, miR-105, miR-106, miR-124,miR-125a, miR-126, miR-130, miR130a, miR-133, miR-142, miR-143, miR-144,miR-145, miR-147, miR-181a, miR-182, miR-183, miR-188, miR-189, miR-192,miR-194, miR-195, miR-199a, miR-200b, miR-201, miR-205, miR-219, 206,miR-215, miR-219, miR-223, miR-224, miR-321, miR-328, miR-331, miR-342.and miR-219, 346. It is contemplated that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 more miRNA (or anyrange derivable therein) may be used for these embodiments. In certainembodiments, methods involve one or more miRNA inhibitors and/or anmiRNA molecules corresponding to any of these miRNAs, particularly forthe treatment or prevention of cancer. Cancer includes, but is notlimited to, malignant cancers, tumors, metastatic cancers, unresectablecancers, chemo- and/or radiation-resistant cancers, and terminalcancers.

In some embodiments of the invention, methods involve one or more miRNAinhibitors and/or an miRNA molecules corresponding to miR-17, miR-21,miR-126, miR-143, miR-145, miR-188, miR-200b, miR-219, or miR-331. Incertain embodiments, methods involve one or more of 1) an inhibitor ofmiR-17, miR-21, miR-182, miR-183, miR-200b, miR-205, miR-223, and/ormiR-224; and/or 2) an miRNA corresponding to let-7, miR-10a, miR-16,miR-22, miR-23, miR-24, miR-26a, miR-29b, miR-30a, miR-106, miR-125a,miR-126, miR-130, miR-133, miR-143, miR-144, miR-145, miR-181a, miR-188,miR-219, miR-192, miR-194, miR-195, miR-199a, mmu-miR-201, miR-215,miR-321, miR-328, miR-331, and/or miR-342. Such methods can be used, insome embodiments to treat cancer, including specific cancers.Additionally, an miRNA corresponding to one or more of miR-15a, miR-16,miR-96, miR-101, miR-105, miR-124, miR-126, miR-142, miR-147, miR-192,miR-194, miR-206, miR-215, or miR-346 may be used to treat cancer orinhibit cell proliferation. It is contemplated that these miRNAs may beused regardless of the source of the cell in which proliferation isundesirable.

It will be understood that shorthand notations are employed such that ageneric description of an miRNA refers to any of its gene family members(distinguished by a number), unless otherwise indicated. It isunderstood by those of skill in the art that a “gene family” refers to agroup of genes having the same miRNA coding sequence. Typically, membersof a gene family are identified by a number following the initialdesignation. For example, miR-16-1 and miR-16-2 are members of themiR-16 gene family and “mir-7” refers to miR-7-1, miR-7-2 and miR-7-3.Moreover, unless otherwise indicated, a shorthand notation refers torelated miRNAs (distinguished by a letter). Thus, “let-7,” for example,refers to let-7a-1, let7-a-2, let-7b, let-7c, let-7d, let-7e, let-7f-1,and let-7f-2.” Exceptions to this shorthand notations will be otherwiseidentified.

The present invention concerns treating breast cancer or decreasing cellproliferation of breast cancer cells by introducing into or providing tothe cell an effective amount of i) an miRNA inhibitor molecule or ii) asynthetic or nonsynthetic miRNA molecule that corresponds to an miRNAsequence. In certain embodiments the methods involves providing aneffective amount of at least 1) one or more miRNA inhibitorscorresponding to miR-21, miR-15a, miR-16, miR-24, and/or miR-25, and/or2) one or more miRNAs corresponding to miR-99, miR-100, miR-205,miR-197, miR-126, miR-143, miR-145 and/or miR-321. Alternatively oradditionally, the miRNAs molecules corresponding to miRNAs (group ii)can include mir-27a, mir-92, mir-96, mir-98, mir-99a, mir-101, mir-105,mir-124, mir-126, mir-129, mir-132, mir-142, mir-147, mir-192, mir-201,mir-206, mir-208, mir-210, mir-211, mir-214, mir-215, mir-219, mir-220,mir-221, mir-223, mir-297, mir-329, mir-331, mir-345, mir-346, mir-409,or mir-411.

It will be understand that the term “providing” an agent is used toinclude “administering” the agent to a patient.

The present invention also relates to treating colon cancer byintroducing into or providing to a colon cancer cell an effective amountof i) an miRNA inhibitor molecule or ii) a synthetic or nonsyntheticmiRNA molecule that corresponds to an miRNA sequence. In certainembodiments the methods involves providing 1) one or more miRNAinhibitors corresponding to miR-21, miR-106, miR-200b, miR-223, miR-224,miR-31, and/or miR-17; and/or 2) one or more miRNAs corresponding tomiR-145, miR-143, miR-133, miR-342, miR-125a, miR-195, miR-30a, miR-10a,miR-130, miR-192, miR-194, miR-215, miR-144, miR-23, miR-26a, miR-126,miR-199a, miR-188, miR-331, and/or miR-21.

Moreover, methods for treating thyroid cancer involve introducing intoor providing to a thyroid cancer cell an effective amount of i) an miRNAinhibitor molecule or ii) a synthetic or nonsynthetic miRNA moleculethat corresponds to an miRNA sequence. In certain embodiments themethods involves providing to the patient 1) one or more miRNAinhibitors corresponding to miR-21 miR-125, miR-24, miR-200b, miR-29b,miR-221, miR-222, miR-224, miR-10a, and/or miR-183; and/or 2) one ormore miRNAs corresponding to miR-145, miR-22, miR-331, miR-126, miR-30a,miR-199a, miR-223, and/or miR-321.

The treatment of lung cancer is also contemplated as part of theinvention. Methods involve introducing into or providing to a lungcancer cell an effective amount of i) an miRNA inhibitor molecule or ii)a synthetic or nonsynthetic miRNA molecule that corresponds to an miRNAsequence. In certain embodiments the methods involves providing to thepatient 1) one or more miRNA inhibitors corresponding to miR-223,miR-106, miR-21, miR-200b, miR-321, miR-182, miR-183, miR-17, and/ormiR-205; and/or 2) one or more miRNAs corresponding to miR-130a,miR-145, miR-126, miR-331, miR-342, miR-143, Let-7, miR-30a, miR-16,miR-26a, miR-125a, miR-29b, miR-24, miR-328, miR-195, miR-22, miR-181a,miR-331, and/or miR-321. Alternatively or additionally, the group ofmiRNA inhibitors (group 1) includes mir-30e-5p, mir-25, mir-32, mir-92,mir-130a, mir-135, mir-145, mir-216, mir-293, mir-294, mir-333, mir-335,mir-338, mir-341, mir-350, mir-369, or mir-412, and the group of miRNAsmolecules corresponding to miRNAs (group 2) includes ambi-mir7100,Let-7b, Let-7d, Let-7g, mir-7, mir-15a, mir-16, mir-22, mir-28, mir-29a,mir-34a, mir-96, mir-101, mir-105, mir-108, mir-122, mir-124, mir-125a,mir-125b, mir-126, mir-128, mir-129, mir-132, mir-133A, mir-136,mir-137, mir-141, mir-142, mir-147, mir-149, mir-151, mir-152, mir-182,mir-183, mir-186, mir-188, mir-192, mir-193, mir-195, mir-223,mir-292-3p, mir-337, mir-337, mir-344, mir-345, mir-346, mir-377, ormir-526b*.

The present invention concerns treating cervical cancer or decreasingcell proliferation of cervical cancer cells by providing an effectiveamount of at least 1) one or more miRNA inhibitors corresponding toLet-7a, Let-7b, Let-7c, Let-7d, Let-7g, mir-9, mir-145, mir-155,mir-181a, mir-186, mir-190, mir-191, or mir-199, and/or 2) one or moremiRNAs corresponding to mir-1, mir-34a, mir-101, mir-124, mir-192,mir-193, mir-195, mir-201, mir-206, mir-208, mir-210, mir-215,mir-292-3p, mir-293, mir-297, mir-299, mir-337, mir-339, mir-340,mir-344, mir-345, mir-367, or mir-409.

The present invention concerns treating prostate cancer or decreasingcell proliferation of prostate cancer cells by introducing into orproviding to the cell an effective amount of i) an miRNA inhibitormolecule or ii) a synthetic or nonsynthetic miRNA molecule thatcorresponds to an miRNA sequence. In certain embodiments the methodsinvolves providing an effective amount of at least 1) one or more miRNAinhibitors corresponding to Let-7a, Let-7b, mir-93, mir-127, mir-154,mir-181a, mir-194, mir-198, mir-199, mir-201, or mir-369, and/or 2) oneor more miRNAs corresponding to mir-15a, mir-16, mir-27a, mir-28,mir-30a-3p, mir-34a, mir-101, mir-103, mir-105, mir-107, mir-124,mir-126, mir-128, mir-129, mir-132, mir-135, mir-137, mir-141, mir-142,mir-147, or mir-297.

The present invention concerns treating skin cancer or decreasing cellproliferation of skin cancer cells by introducing into or providing tothe cell an effective amount of i) an miRNA inhibitor molecule or ii) asynthetic or nonsynthetic miRNA molecule that corresponds to an miRNAsequence. In certain embodiments the methods involves providing aneffective amount of at least 1) one or more miRNA inhibitorscorresponding to miR-26a, miR-125a, miR-128, mir-138, mir-139, mir-141,mir-143, miR-145, mir-146, miR-150, miR-187, mir-188, mir-190, mir-196,mir-197, mir-198, mir-199, miR-201, mir-204, mir-216, miR-223,miR-291-3p, miR-294, miR-295, miR-322, miR-373, mir-410, or mir-412,and/or 2) one or more miRNAs corresponding to let 7a, mir-1, mir-7,mir-15a, mir-16, mir-20, mir-26a, mir-28, mir-34a, mir-96, mir-101,mir-105, miR-105, mir-124, mir-126, mir-128, mir-132, mir-133A, mir-136,mir-137, mir-141, mir-142, mir-144, miR-147, mir-154, mir-181a, mir-192,mir-193, miR-195, mir-201, mir-206, mir-206, mir-215, mir-221, mir-223,mir-291, miR-297, mir-302, miR-324-3p, mir-329, mir-330, miR-337,mir-346, mir-346, mir-373, mu-mir-376b, mir-380-3p, or mir-411. Thepresent invention concerns treating leukemia or decreasing cellproliferation of cancerous T cells by introducing into or providing tothe cell an effective amount of i) an miRNA inhibitor molecule or ii) asynthetic or nonsynthetic miRNA molecule that corresponds to an miRNAsequence. In certain embodiments the methods involves providing aneffective amount of at least 1) one or more miRNA inhibitorscorresponding to miR-15a, miR-23b, miR-25, miR-26a, miR-100, miR-125b,miR-126, miR-129, miR-140, miR-143, or miR-155, and/or 2) one or moremiRNAs corresponding to let-7a, let-7b, miR-10b, miR-17-3p, miR-29a,miR-30a-3p, miR-34a, miR-101, miR-122, or miR-133a. Alternatively oradditionally, the group of miRNA inhibitors (group 1) includes let-7a,let-7b, let-7c, let-7d, let-7g, miR-7, miR-10a, miR-10b, miR-15a,miR-17-3p, miR-18, miR-19a, miR-20, miR-125a, miR-126, or miR-182, andthe group of miRNAs molecules corresponding to miRNAs (group 2) includesmiR-107, miR-134, miR-135, miR-139, miR-141, or miR-145. Moreover, suchmethods can extend to T-cells generally.

In addition to any miRNAs disclosed herein in the context of decreasingcell proliferation, embodiments of the invention include methods fordecreasing cell proliferation comprising introducing into or providingto the cell an effective amount of i) an miRNA inhibitor molecule or ii)a synthetic or nonsynthetic miRNA molecule that corresponds to an miRNAsequence. In certain embodiments the methods involves providing orintroducing an effective amount of at least 1) one or more miRNAinhibitors corresponding to Let-7a, Let-7b, Let-7c, Let-7d, Let-7g,miR-7, mir-9, miR-10a, miR-10b, miR-15a, miR-18, miR-19a, miR-17-3p,miR-20, miR-23b, mir-25, miR-25, miR-26a, miR-26a, mir-30e-5p, mir-32,mir-92, mir-93, miR-100, miR-125a, miR-125b, miR-126, mir-127, miR-128,miR-129, mir-130a, mir-135, mir-138, mir-139, miR-140, mir-141, mir-143,mir-145, mir-146, miR-150, mir-154, mir-155, mir-181a, miR-182, mir-186,miR-187, miR-188, mir-190, mir-191, mir-194, mir-196, mir-197, mir-198,mir-199, mir-201, mir-204, mir-216, miR-223, mir-293, miR-291-3p,miR-294, miR-295, miR-322, mir-333, mir-335, mir-338, mir-341, mir-350,mir-369, miR-373, mir-410, or mir-412, and/or 2) one or more miRNAscorresponding to ambi-mir7100, let 7a, let-7b, let-7d, let-7g, mir-1,mir-7, miR-10b, mir-15a, mir-16, miR-17-3p, mir-20, mir-22, mir-26a,mir-27a, mir-28, mir-28, miR-29a, mir-30a-3p, mir-34a, mir-92, mir-96,mir-98, mir-99a, mir-101, mir-103, mir-105, mir-107, mir-108, mir-122,mir-124, mir-125a, mir-125b, mir-126, mir-128, mir-129, mir-132,miR-133a, miR-134, mir-135, mir-136, mir-137, miR-139, mir-141, mir-142,mir-144, miR-145, mir-147, mir-149, mir-151, mir-152, mir-154, mir-181a,mir-182, mir-183, mir-186, mir-188, mir-192, mir-193, mir-195, mir-195,mir-201, mir-206, mir-208, mir-210, mir-211, mir-214, mir-215, mir-219,mir-220, mir-221, mir-223, mir-291, mir-292-3p, mir-293, mir-297,mir-299, mir-302, miR-324-3p, mir-329, mir-330, mir-331, mir-337,mir-339, mir-340, mir-344, mir-345, mir-346, mir-367, mir-373, miR-376b,mir-377, mir-380-3p, mir-409, mir-411, or mir-526b*. It is particularlycontemplated that such methods may be employed in the context oftreating cancer or another disease or condition in which cellproliferation plays a role, such as hyproliferative diseases andconditions.

The present invention also concerns embodiments methods for increasingcell proliferation comprising introducing into or providing to the cellan effective amount of i) an miRNA inhibitor molecule or ii) a syntheticor nonsynthetic miRNA molecule that corresponds to an miRNA sequence. Incertain embodiments the methods involves providing or introducing aneffective amount of at least 1) one or more miRNA inhibitorscorresponding to ambi-mir71100, let 7a, let-7b, let-7d, let-7g, mir-1,mir-7, miR-10b, mir-15a, mir-16, miR-17-3p, mir-20, mir-22, mir-26a,mir-27a, mir-28, mir-28, miR-29a, mir-30a-3p, mir-34a, mir-92, mir-96,mir-98, mir-99a, mir-101, mir-103, mir-105, mir-107, mir-108, mir-122,mir-124, mir-125a, mir-125b, mir-126, mir-128, mir-129, mir-132,miR-133a, miR-134, mir-135, mir-136, mir-137, miR-139, mir-141, mir-142,mir-144, miR-145, mir-147, mir-149, mir-151, mir-152, mir-154, mir-181a,mir-182, mir-183, mir-186, mir-188, mir-192, mir-193, mir-195, mir-195,mir-201, mir-206, mir-208, mir-210, mir-211, mir-214, mir-215, mir-219,mir-220, mir-221, mir-223, mir-291, mir-292-3p, mir-293, mir-297,mir-299, mir-302, miR-324-3p, mir-329, mir-330, mir-331, mir-337,mir-339, mir-340, mir-344, mir-345, mir-346, mir-367, mir-373, miR-376b,mir-377, mir-380-3p, mir-409, mir-411, or mir-526b*, and/or 2) one ormore miRNAs corresponding to Let-7a, Let-7b, Let-7c, Let-7d, Let-7g,miR-7, mir-9, miR-10a, miR-10b, miR-15a, miR-18, miR-19a, miR-17-3p,miR-20, miR-23b, mir-25, miR-25, miR-26a, miR-26a, mir-30e-5p, mir-32,mir-92, mir-93, miR-100, miR-125a, miR-125b, miR-126, mir-127, miR-128,miR-129, mir-130a, mir-135, mir-138, mir-139, miR-140, mir-141, mir-143,mir-145, mir-146, miR-150, mir-154, mir-155, mir-181a, miR-182, mir-186,miR-187, miR-188, mir-190, mir-191, mir-194, mir-196, mir-197, mir-198,mir-199, mir-201, mir-204, mir-216, miR-223, mir-293, miR-291-3p,miR-294, miR-295, miR-322, mir-333, mir-335, mir-338, mir-341, mir-350,mir-369, miR-373, mir-410, or mir-412. While not limited to such anembodiment, one use for such a method is to increase or induceproliferation of normal cells or other desirable cells in the context ofpretreatment or therapy.

Other aspects of the invention include the treatment of systemic lupuserythrematosus (SLE). In certain embodiments, methods concernintroducing into or providing to a patient an effective amount of i) anmiRNA inhibitor molecule or ii) a synthetic or nonsynthetic miRNAmolecule that corresponds to an miRNA sequence. In certain embodimentsthe methods involves providing to a patient with SLE or suspect ofhaving SLE 1) one or more miRNA inhibitors corresponding to miR-21,miR-223, and/or mir-342 expression; and/or 2) one or more miRNAscorresponding to miR-95, miR-105, miR-137, miR-186, miR-188, miR-199,miR-211, miR-215, mu-miR-290, miR-301, and/or miR-331.

Treatment or prevention of prion diseases is included in methods of theinvention. In some cases, method include introducing into or providingto a patient with a prion disease an effective amount of i) an miRNAinhibitor molecule or ii) a synthetic or nonsynthetic miRNA moleculethat corresponds to an miRNA sequence. In certain embodiments themethods involves providing to a patient 1) one or more miRNA inhibitorscorresponding miR-7, miR-9, miR-16, miR-24, miR-26A, miR-27A, and/ormiR-130A; and/or 2) one or more miRNAs corresponding to miR-95 and/ormiR-135A. The patient may be one diagnosed with a prion disease, one atrisk for a prion disease, or one suspected of having a prion disease. Itis specifically contemplated that in some embodiments of the invention,a nucleic acid molecule corresponding to an miRNA is double stranded,wherein both strands have the sequence of the mature miRNA itcorresponds to. Such a molecule may be designated with an “as” suffix inembodiments of the invention. For example, a nucleic acid moleculecalled miR-9—as was used in some experiments described herein. It iscontemplated that in some embodiments, a nucleic acid molecule is anmiRNA—as molecule.

The present invention also concerns patients diagnosed as havingischemia, those at risk for ischemia, those suspected of havingischemia, or patients with symptoms of ischemia. Methods involveintroducing into or providing to a patient an effective amount of i) anmiRNA inhibitor molecule or ii) a synthetic or nonsynthetic miRNAmolecule that corresponds to an miRNA sequence. In certain embodimentsthe methods involves providing to a patient 1) one or more miRNAinhibitors corresponding to miR-28, miR-30A, miR-31, miR-138, miR-139,miR-140, miR-291 and/or mmu-miR-298; and/or 2) one or more miRNAscorresponding to Let-7f-2 and/or miR-16.

In certain experiments, a synthetic miRNA in which both the sense andantisense strand are derived from a single precursor miRNA is used inmethods and compositions of the invention. These are frequentlydesignated with a “P” suffix in which “5P” indicates that the maturemiRNA derives from the 5′ end of the precursor and a corresponding “3P”indicates that it dferives from the 3′ end of the precursor, asdescribed on the world wide web at sanger.ac.uk/cgi-bin/rfam/mirna.Moreover, in some embodiments, an miRNA that does not correspond to aknown human miRNA was evaluated. It is contemplated that these non-humanmiRNAs may be used in embodiments of the invention or that there mayexist a human miRNA that is homologous to the non-human miRNA.

The present invention in some embodiments concerns methods for reducingcell viability comprising introducing into or providing to the cell aneffective amount of i) an miRNA inhibitor molecule or ii) a synthetic ornonsynthetic miRNA molecule that corresponds to an miRNA sequence. Incertain embodiments the methods involves providing to or introducinginto cells an effective amount of 1) at least one nucleic acid moleculecapable of being processed into a mature miRNA when it is inside thecell, wherein the mature miRNA is let-7a, let-7b, miR-1, miR-10b,miR-17, miR-19a, miR-20, miR-28, miR-29a, miR-30a, miR-32, miR-34a,miR-96, miR-101, miR-122, miR-124, miR-132, miR-133a, miR-134, miR-139,miR-140, miR-144, miR-145, miR-147, miR-155, miR-182, miR-183, miR-184,miR-186, miR-190, miR-193, miR-197, miR-206, miR-208, miR-210, miR-216,miR-217, miR-224, mu-miR-292, mu-miR-293, mu-miR-298, miR-299, miR-301,mu-miR-329, miR-337, mu-miR-344, miR-345, miR-346, miR-369, mu-miR-380,or mu-miR-409; or 2) at least one miRNA inhibitor corresponding tolet-7a, let-7b, let-7c, miR-9, miR-10a, miR-10b, miR-15a, miR-17,miR-18, miR-20, mir-23b, miR-25, miR-26a, miR-98, miR-100, miR-125a,miR-125b, miR-126, miR-129, miR-140, miR-141, miR-143, miR-155, ormiR-181-a. The term “reducing cell viability” means reducing the numberof live cells.

Methods concerning cell viability and cell proliferation may generallybe used for therapeutics, diagnostics, creating cell lines withinteresting research properties, and inducing differentiation. miRNAsthat selectively reduce the proliferation of cancer cells may beemployed as therapeutics since they can be delivered to cancer andnon-cancer cells alike but will only affect the growth of the cancerouscells. In addition, methods may be used to halt or prevent metastasis orreduce the number of metastases.

It is contemplated in some embodiments that the cell in which the effectis desired (referred to as a “targeted cell”), such as a reduction incell viability, may be a cell that is diseased or involved inmaintaining, promoting, or causing a disease or condition. In certainembodiments, the cell is a cancer cell, while in other embodiments, itis contemplated to be a healthy (non-diseased) cell. In certainembodiments, a targeted cell is in an organism.

Moreover, it is particularly contemplated that a nucleic acid moleculecapable of being processed into a mature miRNA when it is inside thecell is a synthetic miRNA in some embodiments of the invention.

In other embodiments, the present invention involves methods forincreasing cell viability comprising introducing into or providing tothe cell an effective amount of i) an miRNA inhibitor molecule or ii) asynthetic or nonsynthetic miRNA molecule that corresponds to an miRNAsequence. In certain embodiments the methods involves providing to orintroducing into cells an effective amount of 1) at least one nucleicacid molecule capable of being processed into a mature miRNA when it isinside the cell, wherein the mature miRNA is let-7a, let-7b, let-7c,miR-9, miR-10a, miR-10b, miR-15a, miR-17, miR-18, miR-20, mir-23b,miR-25, miR-26a, miR-98, miR-100, miR-125a, miR-125b, miR-126, miR-129,miR-140, miR-141, miR-143, miR-155, or miR-181-a; or 2) at least onemiRNA inhibitor corresponding to let-7a, let-7b, miR-1, miR-10b, miR-17,miR-19a, miR-20, miR-28, miR-29a, miR-30a, miR-32, miR-34a, miR-96,miR-101, miR-122, miR-124, miR-132, miR-133a, miR-134, miR-139, miR-140,miR-144, miR-145, miR-147, miR-155, miR-182, miR-183, miR-184, miR-186,miR-190, miR-193, miR-197, miR-206, miR-208, miR-210, miR-216, miR-217,miR-224, mu-miR-292, mu-miR-293, mu-miR-298, miR-299, miR-301,mu-miR-329, miR-337, mu-miR-344, miR-345, miR-346, miR-369, mu-miR-380,or mu-miR-409. The term “increasing cell viability” means that celldeath is inhibited. In particular embodiments, a cancer cell, such as aleukemia cell, is provided with an effective amount of a nucleic acidcapable of being processed into a mature let-7a, let-7b, or miR-10bmolecule.

Methods of the invention also relate to inhibiting cellularproliferation comprising introducing into or providing to the cell aneffective amount of i) an miRNA inhibitor molecule or ii) a synthetic ornonsynthetic miRNA molecule that corresponds to an miRNA sequence. Incertain embodiments the methods involves providing to or introducinginto cells an effective amount of 1) at least one nucleic acid moleculecapable of being processed into a mature miRNA when it is inside thecell, wherein the mature miRNA is let-7a, let-7b, let-7c, let-7d,let-7g, miR-1, miR-7, miR-15a, miR-16, miR-19a, miR-22, miR-28, miR-29a,miR-34a, miR-92, miR-96, miR-98, miR-101, miR-122, miR-124, miR-126,miR-129, miR-133b, miR-137, miR-147, miR-192, miR-193, miR-195, miR-205,miR-206, miR-208, miR-210, mu-miR-292, mu-miR-297, miR-299, miR-337,mu-miR-344, miR-345, or miR-346; or 2) at least one miRNA inhibitorcorresponding to miR-25, miR-27a, miR-31, miR-32, miR-92, miR-139,miR-145, miR-198, miR-212, mu-miR-290, mu-miR-294, miR-323, miR-324,miR-325, miR-331, miR-335, mu-miR-351, miR-369, miR-370, or miR-373.

In some embodiments there are methods of increasing cellularproliferation comprising introducing into or providing to the cell aneffective amount of i) an miRNA inhibitor molecule or ii) a synthetic ornonsynthetic miRNA molecule that corresponds to an miRNA sequence. Incertain embodiments the methods involves providing to or introducinginto cells an effective amount of 1) at least one nucleic acid moleculecapable of being processed into a mature miRNA when it is inside thecell, wherein the mature miRNA is miR-25, miR-27a, miR-31, miR-32,miR-92, miR-139, miR-145, miR-198, miR-212, mu-miR-290, mu-miR-294,miR-323, miR-324, miR-325, miR-331, miR-335, mu-miR-351, miR-369,miR-370, or miR-373; or 2) at least one miRNA inhibitor corresponding tolet-7a, let-7b, let-7c, let-7d, let-7g, miR-1, miR-7, miR-15a, miR-16,miR-19a, miR-22, miR-28, miR-29a, miR-34a, miR-92, miR-96, miR-98,miR-101, miR-122, miR-124, miR-126, miR-129, miR-133b, miR-137, miR-147,miR-192, miR-193, miR-195, miR-205, miR-206, miR-208, miR-210,mu-miR-292, mu-miR-297, miR-299, miR-337, mu-miR-344, miR-345, ormiR-346.

The present invention also covers methods of inhibiting ERK activationintroducing into or providing to a cell an effective amount of i) anmiRNA inhibitor molecule or ii) a synthetic or nonsynthetic miRNAmolecule that corresponds to an miRNA sequence. In certain embodimentsthe methods involves comprising providing to or introducing into cellsan effective amount of one or more miRNA inhibitor corresponding tolet-7a, mir-294, mir-295, miR-19a, miR-25, miR-96, miR-125a, miR-134,miR-148, miR-152, miR-206, miR-207, miR-210, miR-212, miR-216, miR-217,miR-218, miR-223, mu-miR-294, mu-miR-295, miR-301, miR-328, mu-miR-329,miR-339, miR-370, or miR-372.

In certain embodiments, it also covers methods of activating ERK byintroducing into or providing to a cell an effective amount of i) anmiRNA inhibitor molecule or ii) a synthetic or nonsynthetic miRNAmolecule that corresponds to an miRNA sequence. In certain embodimentsthe methods involves providing or introducing into cells an effectiveamount of one or more nucleic acids capable of being processed into amature miRNA when it is inside the cell, wherein the mature miRNA ismiR-19a, miR-25, miR-96, miR-125a, miR-134, miR-148, miR-152, miR-206,miR-207, miR-210, miR-212, miR-216, miR-217, miR-218, miR-223,mu-miR-294, mu-miR-295, miR-301, miR-328, mu-miR-329, miR-339, miR-370,or miR-372. Alternatively or in addition to the mature miRNA is let-7,miR-19a, miR-25, miR-96, miR-125a, miR-134, miR-148, miR-152, miR-206,miR-207, miR-210, miR-212, miR-216, miR-217, miR-218, miR-223,mu-miR-294, mu-miR-295, miR-301, miR-328, mu-miR-329, miR-339, miR-370,or miR-372.

In other embodiments of the invention, there are methods of increasingthe percentage of apoptotic cells in a population comprising introducinginto or providing to the cells an effective amount of i) an miRNAinhibitor molecule or ii) a synthetic or nonsynthetic miRNA moleculethat corresponds to an miRNA sequence. In certain embodiments themethods involves providing to or introducing into cells an effectiveamount of 1) one or more nucleic acid molecules capable of beingprocessed into a mature miRNA when it is inside the cell, wherein themature miRNA is let-7d, miR-22, miR-23a, miR-23b, miR-24, miR-27a,miR-31, miR-128, miR-181a, miR-196, miR-198, miR-199, miR-214, miR-217,mu-miR-290, mu-miR-293, miR-324, miR-338, or mu-miR-412; or 2) an miRNAinhibitor corresponding to miR-34a, miR-96, miR-101, miR-105, miR-126,miR-137, or mu-miR-292. It is specifically contemplated that thepopulation of cells may be diseased or related to a disease orcondition.

In further embodiments of the invention, there are methods of decreasingthe percentage of apoptotic cells in a population comprising introducinginto or providing to the cells an effective amount of i) an miRNAinhibitor molecule or ii) a synthetic or nonsynthetic miRNA moleculethat corresponds to an miRNA sequence. In certain embodiments themethods involves providing to or introducing into cells an effectiveamount of 1) at least one nucleic acid molecule capable of beingprocessed into a mature miRNA when it is inside the cell, wherein themature miRNA is miR-34a, miR-96, miR-101, miR-105, miR-126, miR-137, ormu-miR-292; or 2) at least one miRNA inhibitor corresponding to let-7d,miR-22, miR-23a, miR-23b, miR-24, miR-27a, miR-31, miR-128, miR-181a,miR-196, miR-198, miR-199, miR-214, miR-217, mu-miR-290, mu-miR-293,miR-324, miR-338, or mu-miR-412. It is specifically contemplated thatthe population of cells may be involved in diseases or conditionsinvolving atrophy or the decrease in the number of healthy cells as aresult of apoptosis. One or more of the miRNAs that induce apoptosis maybe introduced into abnormal cells like cancer cells to induce celldeath, providing a therapeutic response. This could be especiallybeneficial if the apoptosis-inducing synthetic miRNAs were injecteddirectly into tumor tissues or otherwise delivered with high efficiencyto primary or metastatic cancer cells. These same miRNAs may beco-delivered with other therapeutic agents like chemotherapies tosupplement their activities and evoke a therapeutic response.Alternatively, the miRNAs that reduce apoptosis may be introduced intonormal cells at the same time that a chemotherapeutic reagent thatinduces apoptosis is introduced, providing some level of protection tothe normal cells while the cancer cells are induced to undergo celldeath. The miRNAs may also be used as targets for diagnostic assays orto differentiate cells or to create cell lines with interesting researchproperties.

Methods of the invention include methods for inhibiting or preventinghTert activity in a cell comprising introducing into or providing to thecell an effective amount of i) an miRNA inhibitor molecule or ii) asynthetic or nonsynthetic miRNA molecule that corresponds to an miRNAsequence. In certain embodiments the methods involves providing to orintroducing into the cell an effective amount of at least one i) miRNAinhibitor corresponding to miR-15a, miR-16, miR-21, mir-24, miR-26a,miR-92, miR-105, miR-125a, miR-125b, miR-128, mir-147, miR-195, miR-207,miR-224, miR-295, mir-301, miR-337, mir-368, or mir-371 or ii) a nucleicacid molecule capable of being processed into a mature miRNA when it isinside the cell, wherein the mature miRNA is miR-26a, miR-147, mir-195,and mir-368. It is specifically contemplated that it is desirable toinhibit hTert activity in cancer cells or in a patient at risk for orsuspected of having cancer. Methods of the invention include methods forinducing hTert activity in a cell comprising providing to or introducinginto the cell an effective amount of at least one nucleic acid moleculecapable of being processed into a mature miRNA when it is inside thecell, wherein the mature miRNA is miR-15a, miR-16, miR-21, mir-24,miR-26a, miR-92, miR-105, miR-125a, miR-125b, miR-128, mir-147, miR-195,miR-207, miR-224, miR-295, mir-301, miR-337, mir-368, or mir-371.Alternatively or additionally, hTert activity may be induced in a cellcomprising providing to or introducing into the cell an miRNA inhibitorcorresponding to miR-26a, miR-147, mir-195, or mir-368.

In other embodiments of the invention there are methods for identifyingan miRNA that inhibits an hTert activating gene product comprising: a)introducing into a cell a candidate miRNA into a cell; and, b) assayingthe level of hTert expression or hTert activity in the cell, wherein areduction in hTert expression or activity compared to a cell lacking themiRNA identifies the miRNA as a potential inhibitor of an hTertactivating gene product. In particular embodiments, the sequence of thecandidate miRNA was previously evaluated for an ability to inhibit anhTert activating gene product. Computer programs and algorithms may beemployed to assess whether a particular miRNA sequence can target aparticular cellular gene. In certain embodiments, thTert activating geneproduct is selected from the group consisting of ACOX1, AKT1, APAF1,COX-5B, COX6, COX7B, CPOX, DUOX2, GPX1, GPX2, GPX4, LPO, MAPK1, MAPK4,MTCO1, NOX3, NOX5, PAOX, PPOX, PRKCA, PRKCD, and TNFRSF6. These methodsmay be used for combating telomerase activity and cancer progression.The invention also includes methods for inhibiting stimulation of Stat3in a cell comprising introducing into or providing to the cell aneffective amount of i) an miRNA inhibitor molecule or ii) a synthetic ornonsynthetic miRNA molecule that corresponds to an miRNA sequence. Incertain embodiments the methods involves providing to the cell aneffective amount of an miRNA selected from the group consisting ofmir-93, mir-100, mir-134, mir-99a, mir-103, mir-128, mir-129, mir-181b,mir-193, mir-197, mir-212, mir-218, mir-219, mir-302, mir-323,mir-324-3p, mir-325, mir-330, mir-331, mir-340, mmu-mir-350, mir-425,mir-491, mir-518f, mir-520a*. Such methods can be used for treatingdiseases and conditions characterized by inflammation. These include,but are not limited to, tissue destruction, organ failure orinflammatory diseases such as Rheumatoid arthritis, Psoriasis, Asthma,Inflammatory bowel disease (Crohn's disease and related conditions),Multiple Sclerosis, obstructive pulmonary disease (COPD), Allergicrhinitis (hay fever), and Cardiovascular disease. Additionally, suchmethods may be used for therapeutics, diagnostics, prognostics, creatingcell lines with interesting research properties, and inducingdifferentiation.

The present invention also concerns methods of influencing the cellcycle of a cell or population of cells. It is contemplated that methodscan involve relatively increasing the number of cells in a particularphase of the cell cycle, such as S, G1, G2/M, or when the number ofchromosomes is greater than 2N. Alternatively, it can involve inducingDNA synthesis in a cell. One or more of the miRNAs involved in the cellcycle can be used to modulate a cell, particularly a cancer cell, toachieve a therapeutic benefit for a patient with such cells. Suchmethods may be used, for example, to enhance the efficacy of atherapeutic agent or they may be employed in the context of research,for instance, to synchronize cells so as to generate a more homogeneouspopulation of cells. Moreover, these miRNAs may regulate genes that areinvolved in controlling cell cycle progression. Mis-expression of one ormore of these miRNAs may profoundly affect the cells in which theyreside, leading potentially toward cancer or other diseases associatedwith altered cell cycle regulation. In addition to using these miRNAs asdiagnostic analytes, they might also provide targets for treatingdisease. For instance, a cancer cell that has bypassed a critical cellcycle signal by having a cell cycle-specific miRNA might be returned tonormalcy by introducing the miRNA.

Methods of promoting cells to be in S phase can be achieved byintroducing into or providing to the cells an effective amount of i) anmiRNA inhibitor molecule or ii) a synthetic or nonsynthetic miRNAmolecule that corresponds to an miRNA sequence. In certain embodimentsthe methods involves providing to or introducing into cells an effectiveamount of 1) at least one nucleic acid molecule capable of beingprocessed into a mature miRNA when it is inside the cell, wherein themature miRNA is let-7a, mir-15a, mir-16, mir-20, mir-26a, mir-191,mir-197, mir-205, mir-220, mir-224, mir-290, mir-291, mir-294, mir-295,mir-302, mir-345, mir-372, or mir-411; or 2) at least one miRNAinhibitor corresponding to mir-108, mir-122, mir-128, mir-129, mir-137,mir-142, mir-146, mir-147, mir-186, mir-187, mir-195, mir-297,mir-324-3p, mir-337, or mir-376b.

The invention also includes methods of inhibiting cells to be in S phaseby introducing into or providing to the cells an effective amount of i)an miRNA inhibitor molecule or ii) a synthetic or nonsynthetic miRNAmolecule that corresponds to an miRNA sequence. In certain embodimentsthe methods involves providing to or introducing into cells an effectiveamount of 1) at least one nucleic acid molecule capable of beingprocessed into a mature miRNA when it is inside the cell, wherein themature miRNA is mir-108, mir-122, mir-128, mir-129, mir-137, mir-142,mir-146, mir-147, mir-186, mir-187, mir-195, mir-297, mir-324-3p,mir-337, or mir-376b; or 2) at least one miRNA inhibitor correspondingto let-7a, mir-15a, mir-16, mir-20, mir-26a, mir-191, mir-197, mir-205,mir-220, mir-224, mir-290, mir-291, mir-294, mir-295, mir-302, mir-345,mir-372, or mir-411.

Methods of promoting cells to be in G1 phase can be achieved byintroducing into or providing to the cells an effective amount of i) anmiRNA inhibitor molecule or ii) a synthetic or nonsynthetic miRNAmolecule that corresponds to an miRNA sequence. In certain embodimentsthe methods involves providing to or introducing into cells an effectiveamount of 1) at least one nucleic acid molecule capable of beingprocessed into a mature miRNA when it is inside the cell, wherein themature miRNA is mir-108, mir-122, mir-124, mir-125a, mir-126, mir-128,mir-129, mir-137, mir-142, mir-146, mir-147, mir-195, mir-201, mir-297,mir-320, mir-325, mir-324-3p, mir-337, mir-371, mir-376b, or mir-409; or2) at least one miRNA inhibitor corresponding to Let-7a, mir-1, mir-7d,mir-20, mir-21, mir-26a, mir-192, mir-193, mir-206, mir-220, mir-290,mir-294, mir-329, mir-371, mir-373, or mir-409.

Other methods concern inhibiting cells in G1 phase by introducing intoor providing to the cells an effective amount of i) an miRNA inhibitormolecule or ii) a synthetic or nonsynthetic miRNA molecule thatcorresponds to an miRNA sequence. In certain embodiments the methodsinvolves providing to or introducing into cells an effective amountof 1) at least one nucleic acid molecule capable of being processed intoa mature miRNA when it is inside the cell, wherein the mature miRNA isLet-7a, mir-1, mir-7d, mir-20, mir-21, mir-26a, mir-192, mir-193,mir-206, mir-220, mir-290, mir-294, mir-329, mir-371, mir-373, mir-409;or 2) at least one miRNA inhibitor corresponding to mir-108, mir-122,mir-124, mir-125a, mir-126, mir-128, mir-129, mir-137, mir-142, mir-146,mir-147, mir-195, mir-201, mir-297, mir-320, mir-325, mir-324-3p,mir-337, mir-371, mir-376b, or mir-409.

Also, there are methods of promoting cells to be in G2/M phase byintroducing into or providing to the cells an effective amount of i) anmiRNA inhibitor molecule or ii) a synthetic or nonsynthetic miRNAmolecule that corresponds to an miRNA sequence. In certain embodimentsthe methods involves providing to or introducing into cells an effectiveamount of 1) at least one nucleic acid molecule capable of beingprocessed into a mature miRNA when it is inside the cell, wherein themature miRNA is mir-1, mir-7a, mir-7d, mir-7g, mir-20, mir-21, mir-26a,mir-145, mir-187, mir-192, mir-193, mir-206, mir-215, mir-220, mir-223,mir-294, mir-329, mir-371, mir-373, or mir-409; or 2) at least one miRNAinhibitor corresponding to mir-15a, mir-18, mir-122, mir-124, mir-126,mir-128, mir-129, mir-137, mir-146, mir-147, mir-195, mir-219, mir-337,or mir-371.

In other embodiments there are methods relating to inhibiting cells tobe in G2/M phase by introducing into or providing to the cells aneffective amount of i) an miRNA inhibitor molecule or ii) a synthetic ornonsynthetic miRNA molecule that corresponds to an miRNA sequence. Incertain embodiments the methods involves providing to or introducinginto cells an effective amount of 1) at least one nucleic acid moleculecapable of being processed into a mature miRNA when it is inside thecell, wherein the mature miRNA is mir-15a, mir-18, mir-122, mir-124,mir-126, mir-128, mir-129, mir-137, mir-146, mir-147, mir-195, mir-219,mir-337, or mir-371; or 2) at least one miRNA inhibitor corresponding tomir-1, mir-7a, mir-7d, mir-7g, mir-20, mir-21, mir-26a, mir-145,mir-187, mir-192, mir-193, mir-206, mir-215, mir-220, mir-223, mir-294,mir-329, mir-371, mir-373, or mir-409.

The present invention also includes methods of increasing the number ofcells with 2× or more DNA in the cell comprising introducing into orproviding to the cells an effective amount of i) an miRNA inhibitormolecule or ii) a synthetic or nonsynthetic miRNA molecule thatcorresponds to an miRNA sequence. In certain embodiments the methodsinvolves providing to or introducing into cells an effective amount ofat least one nucleic acid molecule capable of being processed into amature miRNA when it is inside the cell, wherein the mature miRNA ismiR-1, miR-20, miR-21, miR-337, miR-345, or miR-373. The presentinvention is also concerned with reducing the number of cells with 2×(also referred to as 2N, where N is the number of sets of chromosomes)comprising providing to or introducing into cells an effective amount ofan miRNA inhibitor corresponding to miR-1, miR-20, miR-21, miR-337,miR-345, or miR-373.

In certain embodiments, methods also include targeting an miRNA tomodulate in a cell or organism. The term “targeting an miRNA tomodulate” means a nucleic acid of the invention will be employed so asto modulate the selected miRNA. In some embodiments the modulation isachieved with a synthetic or non-synthetic miRNA that corresponds to thetargeted miRNA, which effectively provides the targeted miRNA to thecell or organism (positive modulation). In other embodiments, themodulation is achieved with an miRNA inhibitor, which effectivelyinhibits the targeted miRNA in the cell or organism (negativemodulation).

In some embodiments, the miRNA targeted to be modulated is an miRNA thataffects a disease, condition, or pathway. In certain embodiments, themiRNA is targeted because a treatment can be provided by negativemodulation of the targeted miRNA. In other embodiments, the miRNA istargeted because a treatment can be provided by positive modulation ofthe targeted miRNA.

In further embodiments of the invention, there is a step of obtaining anucleic molecule of the invention that achieves negative modulation ofthe targeted miRNA. Alternatively, in some cases there is a step ofobtaining a nucleic molecule of the invention that achieves positivemodulation of the targeted miRNA. Thus, it is contemplated that methodsinvolve selecting and/or obtaining a synthetic miRNA, non-syntheticmiRNA or an miRNA inhibitor (collectively “miRNA modulators”) thatcorresponds to a targeted miRNA, such as one that is involved with,affects or is characteristic of a particular disease, condition,pathway, or factor in the pathway.

In certain methods of the invention, there is a further step ofadministering the selected miRNA modulator to a cell, tissue, organ, ororganism (collectively “biological matter”) in need of treatment relatedto modulation of the targeted miRNA or in need of the physiological orbiological results discussed herein (such as with respect to aparticular cellular pathway or result like decrease in cell viability).Consequently, in some methods of the invention there is a step ofidentifying a patient in need of treatment that can be provided by themiRNA modulator(s). It is contemplated that an effective amount of anmiRNA modulator can be administered in some embodiments. In particularembodiments, there is a therapeutic benefit conferred on the biologicalmatter, where a “therapeutic benefit” refers to an improvement in theone or more conditions or symptoms associated with a disease orcondition or an improvement in the prognosis, duration, or status withrespect to the disease. It is contemplated that a therapeutic benefitincludes, but is not limited to, a decrease in pain, a decrease inmorbidity, a decrease in a symptom. For example, with respect to cancer,it is contemplated that a therapeutic benefit can be inhibition of tumorgrowth, prevention of metastasis, reduction in number of metastases,inhibition of cancer cell proliferation, inhibition of cancer cellproliferation, induction of cell death in cancer cells, inhibition ofangiogenesis near cancer cells, induction of apoptosis of cancer cells,reduction in pain, reduction in risk of recurrence, induction of chemo-or radiosensitivity in cancer cells, prolongation of life, and/or delayof death directly or indirectly related to cancer.

It is specifically contemplated that miRNA profiles for patients,particularly those suspected of having a particular disease orcondition, can be generated by evaluating any of the miRNAs discussed inthis application. The miRNA profile that is generated from the patientwill be one that provides information regarding the particular diseaseor condition. In many embodiments, the miRNA profile is generated usingthe miRNA array discussed.

Furthermore, it is contemplated that the miRNA compositions may beprovided as part of a therapy to a patient, in conjunction withtraditional therapies or preventative agents. Moreover, it iscontemplated that any method discussed in the context of therapy may beapplied as preventatively, particularly in a patient identified to bepotentially in need of the therapy or at risk of the condition ordisease for which a therapy is needed.

In other embodiments, the invention concerns a method for inducingtransformation in a cell comprising administering to the cell aneffective amount of at least one miRNA selected from the groupconsisting of mir-192, mir-198, and mir-199. Alternatively, methods forpreventing cell transformation may be achieved by administering to thecell an effective amount of at least one miRNA inhibitor of mir-192,mir-198, or mir-199.

In addition, methods of the invention concern employing one or morenucleic acids corresponding to an miRNA and a therapeutic drug. Thenucleic acid can enhance the effect or efficacy of the drug, reduce anyside effects or toxicity, modify its bioavailability, and/or decreasethe dosage or frequency needed. In certain embodiments, the therapeuticdrug is a cancer therapeutic. Consequently, in some embodiments, thereis a method of treating cancer in a patient comprising administering tothe patient the cancer therapeutic and an effective amount of at leastone miRNA molecule that improves the efficacy of the cancer therapeuticor protects non-cancer cells. Furthermore, in some cases the miRNAmolecule enhances the efficacy of the cancer therapeutic and is selectedfrom the group consisting of ambi-miR-7100, mir-28, mir-101, mir-124,mir-125a, mir-126, mir-132, mir-136, mir-147, mir-155, mir-182, mir-186,mir-202, mir-206, mir-216, mir-221, mir-224, mir-291, mir-292-3p,mir-297, mir-302, mir-337, mir-372, mir-373, and mir-376b.

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude but are not limited to, for example, bevacizumab, cisplatin(CDDP), carboplatin, EGFR inhibitors (gefitinib and cetuximab),procarbazine, mechlorethamine, cyclophosphamide, camptothecin, COX-2inhibitors (e.g., celecoxib) ifosfamide, melphalan, chlorambucil,busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin(adriamycin), bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,taxotere, gemcitabien, navelbine, farnesyl-protein transferaseinhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin andmethotrexate, or any analog or derivative variant of the foregoing.

Alternatively or additionally, the miRNA molecule in methods of theinvention protects non-cancer cells from the cancer therapeutic and isselected from the group consisting of mir-16, mir-24, mir-30a-3p,mir-125b, mir-152, mir-194, mir-197, mir-214, and mir-331.

Generally, inhibitors of miRNAs can be given to achieve the oppositeeffect as compared to when nucleic acid molecules corresponding to themature miRNA are given. Similarly, nucleic acid molecules correspondingto the mature miRNA can be given to achieve the opposite effect ascompared to when inhibitors of the miRNA are given. For example, miRNAmolecules that increase cell proliferation can be provided to cells toincrease proliferation or inhibitors of such molecules can be providedto cells to decrease cell proliferation. The present inventioncontemplates these embodiments in the context of the differentphysiological effects observed with the different miRNA molecules andmiRNA inhibitors disclosed herein. These include, but are not limitedto, the following physiological effects: increase and decreasing cellproliferation, increasing or decreasing apoptosis, increasingtransformation, increasing or decreasing cell viability, activating ERK,activating/inducing or inhibiting hTert, inhibit stimulation of Stat3,reduce or increase viable cell number, and increase or decrease numberof cells at a particular phase of the cell cycle. Methods of theinvention are generally contemplated to include providing or introducingone or more different nucleic acid molecules corresponding to one ormore different miRNA molecules. It is contemplated that the following,at least the following, or at most the following number of differentnucleic acid molecules may be provided or introduced: 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, or any range derivable therein. This also applies to thenumber of different miRNA molecules that can be provided or introducedinto a cell.

The present invention also concerns kit containing compositions of theinvention or compositions to implement methods of the invention. In someembodiments, kits can be used to evaluate one or more miRNA molecules.In certain embodiments, a kit contains, contains at least or contains atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more syntheticmiRNA molecules or miRNA inhibitors, or any range and combinationderivable therein. In some embodiments, there are kits for evaluatingmiRNA activity in a cell.

Kits may comprise components, which may be individually packaged orplaced in a container, such as a tube, bottle, vial, syringe, or othersuitable container means.

Individual components may also be provided in a kit in concentratedamounts; in some embodiments, a component is provided individually inthe same concentration as it would be in a solution with othercomponents. Concentrations of components may be provided as 1×, 2×, 5×,10×, or 20× or more.

Kits for using synthetic miRNAs, nonsynthetic, and/or miRNA inhibitorsof the invention for therapeutic, prognostic, or diagnostic applicationsare included as part of the invention. Specifically contemplated are anysuch molecules corresponding to any miRNA reported to influencebiological activity, such as those discussed herein.

Negative and/or Positive Control synthetic miRNAs and/or miRNAinhibitors are included in some kit embodiments. The Control moleculescan be used to verify transfection efficiency and/or control fortransfection-induced changes in cells.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein and that different embodiments may be combined. It isspecifically contemplated that any methods and compositions discussedherein with respect to miRNA molecules or miRNA may be implemented withrespect to synthetic miRNAs to the extent the synthetic miRNA is exposedto the proper conditions to allow it to become a mature miRNA underphysiological circumstances. The claims originally filed arecontemplated to cover claims that are multiply dependent on any filedclaim or combination of filed claims.

Any embodiment of the invention involving specific miRNAs by name iscontemplated also to cover embodiments involving miRNAs whose sequencesare at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99% identical to the mature sequence of the specifiedmiRNA.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is specifically contemplated that any embodiments described in theExamples section are included as an embodiment of the invention.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 Overview of miRNA Expression and Activation. mRNAs aretranscribed as part of longer RNA molecules that can be as long as athousand nucleotides (Lee, 2002). The RNAs are processed in the nucleusinto hairpin RNAs of 70-100 nucleotides by the dsRNA-specificribonuclease Drosha (Lee 2003) (FIG. 1). The hairpin RNAs aretransported to the cytoplasm and digested by a second, double-strandspecific ribonuclease called Dicer. The resulting 19-23mer miRNA isbound by a complex that is similar to or identical to the RNA-InducedSilencing Complex (RISC) that participates in RNA interference(Hutvagner, 2002). The complex-bound, single-stranded miRNA binds mRNAswith sequences that are significantly, though not completely,complementary to the miRNA. By a mechanism that is not fully understood,but that does not involve mRNA degradation, the bound mRNA is nottranslated, resulting in reduced expression of the corresponding gene.

FIG. 2. Methods for Introducing miRNAs into Cells. There are three basicmethods for introducing miRNAs into cells. In the first, a DNA bearing apromoter upstream of a sequence encoding a miRNAs is introduced intocells where it is transcribed to produce an RNA molecule that includesthe mature miRNA. Processing and uptake by the protein complex formiRNA-induced gene regulation results in the activation of the miRNA.This method suffers from inefficient introduction of the DNA constructinto cells. In the second method, an siRNA-like dsRNA molecule, one ofwhose strands is identical to an active miRNA is introduced into cellswhere it is taken up by the protein complex for miRNA activation. Thismethod provides efficient deliver, but often uptake of the unintendedcomplementary RNA molecule. The third method, described herein, involvesmodifying the complementary strand so as to favor uptake and activationof the active strand of the synthetic miRNA construct.

FIG. 3. Preferential Uptake of Active Strands in synthetic miRNAs of theinvention. Reporter vectors with luciferase under the control of targetsites for miR-33 or let-7 or the complementary strands of theafore-mentioned siRNAs. Co-transfection of synthetic miRNAs and reportervectors followed by luciferase assay 24 hours post-transfection revealedmiRNAs that are activated following transfection.

FIG. 4. Synthetic miRNA Activity for various miRNAs. Synthetic miRNAswith siRNA and Pre-miR (5′amine) design were prepared and transfectedinto HeLa cells at 3 and 10 nM final concentration. The synthetic miRNAswere co-transfected with reporter vectors bearing target sites for themature miRNAs. The expression of the luciferase reporter inco-transfected cells was measured twenty-four hours post-transfected andexpressed in the figure as the reporter expression relative to cellsco-transfeted with negative control synthetic miRNAs.

FIG. 5. Synthetic miRNA Activity across Cell Types and Against NaturalTargets. Synthetic miRNAs were tested for proper strand activation andcell-type specificity to ensure that the design is robust. Fourdifferent cell types were co-transfected with synthetic miRNA andassociated active and complementary strand activation. Panel A showsthat different cell types respond similarly to synthetic miRNAs. Fourdifferent synthetic miRNAs were then transfected into various cell typesand the expression levels of natural targets of the miRNAs were measured(Panel B).

FIG. 6. Schematic for screening with libraries of synthetic miRNAs ormiRNA inhibitors. Synthetic miRNAs and/or miRNA inhibitors aredistributed to wells of a microtiter plate. Transfection reagent andthen cells are added to each well. At some time post-transfection,samples are evaluated for a phenotype. MiRNAs that induce a change thatis significant relative to a negative control are selected for furtherstudy.

FIG. 7. Screen for miRNAs that affect cell proliferation. In 96-wellplates, 8,000 HeLa cells were reverse transfected with miRNA inhibitors(5 pmoles) in triplicates using Ambion siPORT Neo-FX. 72 hourspost-transfection, cells were fixed with 4% paraformaldehyde,permiabilized with 0.1% TritonX 100 and stained with propidium iodide tolook at total cell number. The plates were scanned using the TTP labtechAcumen Explorer. Morphology changes in cells inhibited for mir 31. HeLacells were transfected with Anti-mir31 and cells were fixed and stainedwith anti-beta actin antibody and DAPI to visualize cell morphologychanges in response to inhibition to mir-31 micro-RNA function.

FIG. 8. Screen for miRNAs that affect cell proliferation in A549 cells.Screen for miRNA involved in cell viability in A549 cells. In 96-wellplates, 8,000 A549 cells were reverse transfected with miRNA inhibitors(5 pmoles) in triplicates using Ambion siPORT Neo-FX. 72 hourspost-transfection cells were trypsinized and counted using the Guavacell counting instrument. Cell number was graphed and normalized to agap inhibitor. In this figure, “mir1d” refers to mir-1-2.

FIG. 9. Screen for miRNAs that affect apoptosis in HeLa cells. Effectsof miRNA inhibitors on caspase activity in HeLa. In 96-well plates,8,000 HeLa cells were reverse transfected with miRNA inhibitors (5pmoles) in triplicates using Ambion siPORT Neo-FX. 72 hourspost-transfection cells were analyzed using caspase activity assay andnormalized based on esterase activity assay. In this figure, “mir1d”refers to mir-1-2.

FIG. 10. miRNA Expression in Lung and Colon Cancer Patients. The miRNAexpression profiles of tumor vs normal adjacent tissues were comparedfor lung and colon cancer patients. The miRNAs are provided in rows; thepatients are presented in columns. Green in the heat map shows miRNAsthat are down-regulated in the tumor sample relative to the normaladjacent tissue sample, and red shows miRNAs that are up-regulated inthe tumor sample relative to the normal adjacent tissue sample.

FIG. 11. Validation of miRNA Array Expression Results in Lung CancerPatients. Total RNA samples from two lung cancer patients were analyzedfor expression of miR-16, miR-21, miR-143, miR-145, and let-7 usingNorthern analysis. The graphs show the relative abundance of each miRNA(ratio of tumor:NAT) from the array analysis and Northern phosphoimageranalysis.

FIG. 12. Some miRNAs are differentially expressed in multiple cancertypes. miRNA array analysis comparing tumor and normal adjacent tissuesfrom patients with various types of cancer was used to identify miRNAsthat are differentially expressed in cancer. The percentage of patientsexhibiting up- or down-regulation of a given miRNA was calculated foreach cancer type. The eight that were most often differentiallyexpressed across sample types are presented.

FIG. 13. Shown are miRNAs having greater than 1.5-fold expressionchanges between both infected vs. uninfected and sensitive vs.insensitive. On the right is a cluster of the results from 2 arrays ofeach model.

FIG. 14. Differentially expressed miRNAs in 3 preconditioned micerelative to non-treated mice.

FIG. 15A-C. Synthetic miRNAs that decrease cell proliferation. A. BT549and MCF12A (breast), HeLa (cervical) and 22 Rv1 (prostate) cells wereevaluated for cell proliferation. B. TE354T and TE353SK (skin), BJ(skin), and A549 (lung) cells were examined for cell proliferation. C.CRL5826 and HTB-57 (lung), Jurkats (T cell), and primary T cells wereevaluated for cell proliferation.

FIG. 16. Synthetic miRNAs that increase cell proliferation. HeLa(cervical), 22 Rv1 (prostate), TE354T and TE353SK (skin), BJ (skin),A549 (lung), Jurkats (T cell), primary T cells, CRL5826 and HTB-57(lung) cells were evaluated for cell proliferation.

FIG. 17. miRNA inhibitors that reduce cell proliferation. 22 Rv1(prostate), TE354T (skin), MCF12a (breast), and A549 (lung) cells wereevaluated for cell proliferation.

FIG. 18. miRNA inhibitors that increase cell proliferation. 22 Rv1(prostate), TE354T (skin), MCF12a (breast), and A549 (lung) cells wereevaluated for cell proliferation.

FIG. 19. miRNAs that affect cell viability. Jurkats (T cell), primary Tcells, HeLa (cervical) and A549 (lung) cells were evaluated forincreases and decreases in cell viability.

FIG. 20. miRNAs that affect apoptosis. 22 Rv1 (prostate), TE354T (skin),Jurkats (T cell), and HeLa (cervical) cells were evaluated for increasesand decreases in apoptosis.

FIG. 21. miRNAs that affect cell viability in the presence of atherapeutic. A549 (lung) cells were evaluated for increases anddecreases in cell viability in the presence and absence of TRAIL oretoposide. HTB-57 and CRL5826 (lung) and HeLa (cervical) cells wereevaluated for a reduction in cell viability in the absence and presenceof etoposide.

FIG. 22. miRNAs that affect cell cycle. BJ (skin) and HeLa (cervical)cells were evaluated for increases or decreases in the number of cellsat certain phases of the cell cycle (G1, S, G2/M, DNA replication).

FIG. 23. Phenotypes of miRNAs with similar sequences. Comparison ofrelated sequences and their effects on cell proliferation.

FIG. 24. Genes associated with hTert regulation and miRNA sequencespredicted to modulate their expression.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is directed to compositions and methods relatingto preparation and characterization of miRNAs, as well as use of miRNAsfor therapeutic, prognostic, and diagnostic applications. To overcomethe problem with previous inefficient plasmid-based systems forintroducing miRNA into cells, the inventors developed small, partiallydouble-stranded RNAs that can be delivered with high efficiency to bothimmortalized and primary cells. The small RNAs have the same functionalactivities as endogenously expressed miRNAs. Because the small RNAs canbe delivered to cells with much higher efficiency than can plasmids,they induce a much stronger phenotype that is easier to detect andquantify, making it possible to identify many of the functions of miRNAsin cells.

The inventors have also created a library of the small, double-strandedRNA molecules that can be used to introduce miRNAs into cells, as wellas a library of antisense molecules that inhibit the activities of knownmiRNAs that are present in cells. These libraries have been used tosequentially up- or down-regulate one or more miRNAs in cells toidentify those miRNAs that are critical for cellular processes like cellcycle, apoptosis, differentiation, viability, angiogenesis, metabolism,and other processes with therapeutic potential. miRNAs that regulate theexpression of important genes like p53, MYC, and RAS are also beingidentified and characterized to further pinpoint miRNAs that mightprovide important intervention points for treating disease. For example,let-7 has been shown to be involved with RAS. See Johnson et al., 2005,which is hereby incorporated by reference. These processes of seriallymodulating miRNA activities and assaying for cellular phenotypes arecollectively referred to as miRNA functional screening.

I. miRNA Molecules

MicroRNA molecules (“miRNAs”) are generally 21 to 22 nucleotides inlength, though lengths of 17 and up to 25 nucleotides have beenreported. The miRNAs are each processed from a longer precursor RNAmolecule (“precursor miRNA”). Precursor miRNAs are transcribed fromnon-protein-encoding genes. The precursor miRNAs have two regions ofcomplementarity that enables them to form a stem-loop- or fold-back-likestructure, which is cleaved by an enzyme called Dicer in animals. Diceris ribonuclease III-like nuclease. The processed miRNA is typically aportion of the stem.

The processed miRNA (also referred to as “mature miRNA”) become part ofa large complex to down-regulate a particular target gene. Examples ofanimal miRNAs include those that imperfectly basepair with the target,which halts translation (Olsen et al., 1999; Seggerson et al., 2002).SiRNA molecules also are processed by Dicer, but from a long,double-stranded RNA molecule. SiRNAs are not naturally found in animalcells, but they can function in such cells in a RNA-induced silencingcomplex (RISC) to direct the sequence-specific cleavage of an mRNAtarget (Denli et al., 2003).

The study of endogenous miRNA molecules is described in U.S. PatentApplication 60/575,743, which is hereby incorporated by reference in itsentirety.

Synthetic miRNAs

miRNAs are apparently active in the cell when the mature,single-stranded RNA is bound by a protein complex that regulates thetranslation of mRNAs that hybridize to the miRNA. Introducing exogenousRNA molecules that affect cells in the same way as endogenouslyexpressed miRNAs requires that a single-stranded RNA molecule of thesame sequence as the endogenous mature miRNA be taken up by the proteincomplex that facilitates translational control. A variety of RNAmolecule designs have been evaluated. Three general designs thatmaximize uptake of the desired single-stranded miRNA by the miRNApathway have been identified. An RNA molecule with an miRNA sequencehaving at least one of the three designs is referred to as a syntheticmiRNA.

Synthetic miRNAs of the invention comprise, in some embodiments, two RNAmolecules wherein one RNA is identical to a naturally occurring, maturemiRNA. The RNA molecule that is identical to a mature miRNA is referredto as the active strand. The second RNA molecule, referred to as thecomplementary strand, is at least partially complementary to the activestrand. The active and complementary strands are hybridized to create adouble-stranded RNA, called the synthetic miRNA, that is similar to thenaturally occurring miRNA precursor that is bound by the protein compleximmediately prior to miRNA activation in the cell. Maximizing activityof the synthetic miRNA requires maximizing uptake of the active strandand minimizing uptake of the complementary strand by the miRNA proteincomplex that regulates gene expression at the level of translation. Themolecular designs that provide optimal miRNA activity involvemodifications to the complementary strand.

Two designs incorporate chemical modifications in the complementarystrand. The first modification involves creating a complementary RNAwith a chemical group other than a phosphate or hydroxyl at its 5′terminus. The presence of the 5′ modification apparently eliminatesuptake of the complementary strand and subsequently favors uptake of theactive strand by the miRNA protein complex. The 5′ modification can beany of a variety of molecules including NH₂, NHCOCH₃, biotin, andothers.

The second chemical modification strategy that significantly reducesuptake of the complementary strand by the miRNA pathway is incorporatingnucleotides with sugar modifications in the first 2-6 nucleotides of thecomplementary strand. It should be noted that the sugar modificationsconsistent with the second design strategy can be coupled with 5′terminal modifications consistent with the first design strategy tofurther enhance synthetic miRNA activities.

The third synthetic miRNA design involves incorporating nucleotides inthe 3′ end of the complementary strand that are not complementary to theactive strand. Hybrids of the resulting active and complementary RNAsare very stable at the 3′ end of the active strand but relativelyunstable at the 5′ end of the active strand. Studies with siRNAsindicate that 5′ hybrid stability is a key indicator of RNA uptake bythe protein complex that supports RNA interference, which is at leastrelated to the miRNA pathway in cells. The inventors have found that thejudicious use of mismatches in the complementary RNA strandsignificantly enhances the activity of the synthetic miRNA.

MiRNA Libraries

A key application for the synthetic miRNAs is the identification ofcellular functions for individual or groups of miRNAs. The inventorshave created a library of synthetic miRNAs that can be used tosequentially introduce each of the known miRNAs into cultured cells(FIG. 6). Cell populations with each of the different synthetic miRNAscan then be assayed to identify miRNAs whose presence induces a cellularphenotype.

The inventors have created a library of antisense molecules that inhibitmiRNA activity. The miRNA inhibitors are used to serially inhibit theactivities of miRNAs in cells to identify miRNAs whose absence induces acellular phenotype.

The number of different synthetic miRNAs or miRNA inhibitors in thelibraries is variable. It is contemplated that there may be, be atleast, or be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 31,3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300,4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000or more, or any range derivable therein, different miRNA-specificmolecules in the library. In specific embodiments, libraries havebetween 5 and 1000 different miRNA-specific molecules, between 20 and500 different miRNA-specific molecules, between 50 and 250 differentmiRNA-specific molecules, or between 100 and 225 differentmiRNA-specific molecules. “Different” miRNA-specific molecules refers tonucleic acids that are specific to miRNAs with different sequences.

Synthetic miRNAs are contemplated to be made primarily of RNA, though insome embodiments, they may be RNA, nucleotide analogs, DNA, or anycombination of DNA, RNA, nucleotide analogs, and PNAs.

As suggested above, it is contemplated that libraries of the inventionmay be specific for one or more miRNAs. In embodiments of the invention,a library has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700,710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840,850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980,990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 31, 3200,3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400,4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 ormore, or any range derivable therein, different miRNAs or miRNAinhibitors. Accordingly, it is understood that the library contains oneor more nucleic acids for these different miRNAs. In specificembodiments, the library is specific to human miRNAs, though librariesfor multiple organisms are contemplated.

RNA molecules of the invention have miRNA regions or complementaryregions. In specific embodiments, a synthetic miRNA or miRNA inhibitorhas a sequence or complementary sequence that derives from any of SEQ IDNOs: 1-805, inclusive. It is particularly contemplated that syntheticnucleic acid molecules of the invention may be derived from any of themature miRNA sequences in SEQ ID NOs:1-805 or their complement.

As discussed above, miRNAs are processed from a precursor molecule. Incertain embodiments, the specific length of a mature miRNA is unknown.It is contemplated that versions of the synthetic miRNA and miRNAinhibitor libraries will include sequence that extends at least 1 to 5nucleotides of coding sequence upstream and/or downstream of thepredicted miRNA sequence. In some embodiments, molecules have up to 1,2, 3, 4, 5, 6, 7, or more contiguous nucleotides, or any range derivabletherein, that flank the sequence encoding the predominant processedmiRNA on one or both sides (5′ and/or 3′ end).

The present invention concerns methods for creating functional profilefor all of the known miRNAs. The term “functional profile” refers to aset of data regarding the cellular phenotypes that result fromintroducing and inhibiting miRNAs in cells using synthetic miRNA andmiRNA inihibitor libraries. Functional profiles for individual miRNAswill enable identification of miRNAs with therapeutic or diagnosticpotential. For instance, a functional profile for a miRNA might revealthat its absence leads to uncontrolled cell proliferation and aninability to induce apoptosis following DNA damage. Furthermore, theexpression of p53 correlates with whether the miRNA is beingup-regulated with a synthetic miRNA or down-regulated with a miRNAinhibitor. Based on its ties to cell proliferation, apoptosis, and p53expression, this miRNA might be a target for cancer therapeutics.

In certain embodiments, methods concern identifying miRNAs indicative ofa disease or condition by detecting a correlation between the activityof particular miRNAs and cellular phenotypes that coincide with adisease or condition.

Libraries of the invention can contain miRNA sequences from any organismhaving miRNAs, specifically including but not limited to, mammals suchas humans, mice, and rats. Specifically contemplated are librarieshaving, having at least, or having at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295,296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337,338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365,366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379,380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393,394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407,408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421,422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435,436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463,464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477,478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491,492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505,506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519,520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547,548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561,562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200,3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400,4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600,5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800,6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000,8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200,9300, 9400, 9500, 9600, 9700, 9800, 9900, 10000 or more differentsynthetic miRNAs and/or miRNA inhibitors (that is, miRNA-specificmolecules having different sequences derived from different miRNAgenes). Specifically contemplated are such libraries described in theprevious sentence with respect to any of SEQ ID NOs:1-805, particularlythose corresponding to miRNA sequence s(mature sequence) or thecomplement thereof.

A. Nucleic Acids

The present invention concerns nucleic acid molecules that can introduceor inhibit miRNAs in cultured cells. The nucleic acids may have beenproduced in cells or in vitro by purified enzymes though they arepreferentially produced by chemical synthesis. They may be crude orpurified. The term “miRNA,” unless otherwise indicated, refers to theprocessed RNA, after it has been cleaved from its precursor. Table 1indicates which SEQ ID NO corresponds to the particular precursorsequence of an miRNA and what sequences within the SEQ ID NO correspondto the mature sequence. The name of the miRNA is often abbreviated andreferred to without the prefix and will be understood as such, dependingon the context. Unless otherwise indicated, miRNAs referred to in theapplication are human sequences identified as mir-X or let-X, where X isa number and/or letter.

TABLE 1 Human miRNA Sequences Processed Sequence miRNA name PrecursorRelative to Precursor hsa-mir-1-2 SEQ ID NO: 1 53-73 hsa-mir-1-1 SEQ IDNO: 2 46-66 hsa-let-7a-1 SEQ ID NO: 3 6-27 hsa-let-7a-2 SEQ ID NO: 45-26 hsa-let-7a-3 SEQ ID NO: 5 4-25 hsa-let-7b SEQ ID NO: 6 6-27hsa-let-7c SEQ ID NO: 7 11-32 hsa-let-7d SEQ ID NO: 8 8-28 hsa-let-7eSEQ ID NO: 9 8-28 hsa-let-7f-1 SEQ ID NO: 10 7-28 hsa-let-7f-2 SEQ IDNO: 11 8-29 hsa-mir-7-1 SEQ ID NO: 12 24-44 hsa-mir-7-2 SEQ ID NO: 1332-52 hsa-mir-7-3 SEQ ID NO: 14 31-51 hsa-let-7g SEQ ID NO: 15 5-25hsa-let-7i SEQ ID NO: 16 6-24 hsa-mir-9-1 SEQ ID NO: 17 16-38 and/or56-76 hsa-mir-9-2 SEQ ID NO: 18 16-38 and/or 54-74 hsa-mir-9-3 SEQ IDNO: 19 16-38 and/or 56-76 hsa-mir-10a SEQ ID NO: 20 22-44 hsa-mir-10bSEQ ID NO: 21 27-48 hsa-mir-15a SEQ ID NO: 22 14-35 hsa-mir-15b SEQ IDNO: 23 20-41 hsa-mir-16-1 SEQ ID NO: 24 14-35 hsa-mir-16-2 SEQ ID NO: 2510-31 hsa-mir-17 SEQ ID NO: 26 14-37 and/or 51-70 hsa-mir-18 SEQ ID NO:27 6-27 hsa-mir-19a SEQ ID NO: 28 49-71 hsa-mir-19b-1 SEQ ID NO: 2954-76 hsa-mir-19b-2 SEQ ID NO: 30 62-84 hsa-mir-20 SEQ ID NO: 31 8-29hsa-mir-21 SEQ ID NO: 32 8-29 hsa-mir-22 SEQ ID NO: 33 53-74 hsa-mir-23aSEQ ID NO: 34 45-65 hsa-mir-23b SEQ ID NO: 35 58-80 hsa-mir-24-1 SEQ IDNO: 36 6-28 and/or 44-65 hsa-mir-24-2 SEQ ID NO: 37 50-71 hsa-mir-25 SEQID NO: 38 52-73 hsa-mir-26a-1 SEQ ID NO: 39 10-31 hsa-mir-26b SEQ ID NO:40 12-32 hsa-mir-26a-2 SEQ ID NO: 41 14-35 hsa-mir-27a SEQ ID NO: 4251-72 hsa-mir-27b SEQ ID NO: 43 61-80 hsa-mir-28 SEQ ID NO: 44 14-35hsa-mir-29a SEQ ID NO: 45 41-62 hsa-mir-29b-1 SEQ ID NO: 46 51-70hsa-mir-29b-2 SEQ ID NO: 47 52-71 hsa-mir-29c SEQ ID NO: 48 54-75hsa-mir-30a SEQ ID NO: 49 47-68 hsa-mir-30c-2 SEQ ID NO: 50 7-29hsa-mir-30d SEQ ID NO: 51 6-27 hsa-mir-30b SEQ ID NO: 52 17-37hsa-mir-30c-1 SEQ ID NO: 53 17-39 hsa-mir-30e SEQ ID NO: 54 2-21hsa-mir-31 SEQ ID NO: 55 9-29 hsa-mir-32 SEQ ID NO: 56 6-26 hsa-mir-33SEQ ID NO: 57 6-24 hsa-mir-34a SEQ ID NO: 58 22-43 hsa-mir-34b SEQ IDNO: 59 14-35 hsa-mir-34c SEQ ID NO: 60 13-34 hsa-mir-92-1 SEQ ID NO: 6148-69 hsa-mir-92-2 SEQ ID NO: 62 48-69 hsa-mir-93 SEQ ID NO: 63 12-33hsa-mir-95 SEQ ID NO: 64 49-70 hsa-mir-96 SEQ ID NO: 65 9-30 hsa-mir-98SEQ ID NO: 66 2-23 hsa-mir-99a SEQ ID NO: 67 13-34 hsa-mir-99b SEQ IDNO: 68 7-28 hsa-mir-100 SEQ ID NO: 69 13-34 hsa-mir-101-1 SEQ ID NO: 7047-68 hsa-mir-101-2 SEQ ID NO: 71 49-70 hsa-mir-103-2 SEQ ID NO: 7248-70 hsa-mir-103-1 SEQ ID NO: 73 48-70 hsa-mir-105-1 SEQ ID NO: 7413-32 hsa-mir-105-2 SEQ ID NO: 75 13-32 hsa-mir-106a SEQ ID NO: 76 13-36hsa-mir-106b SEQ ID NO: 77 12-32 hsa-mir-107 SEQ ID NO: 78 50-72hsa-mir-122a SEQ ID NO: 79 15-37 hsa-mir-124a-1 SEQ ID NO: 80 52-73hsa-mir-124a-2 SEQ ID NO: 81 61-82 hsa-mir-124a-3 SEQ ID NO: 82 52-73hsa-mir-125b-1 SEQ ID NO: 83 15-36 hsa-mir-125a SEQ ID NO: 84 15-37hsa-mir-125b-2 SEQ ID NO: 85 17-38 hsa-mir-126 SEQ ID NO: 86 15-35and/or 52-72 hsa-mir-127 SEQ ID NO: 87 57-78 hsa-mir-128a SEQ ID NO: 8850-71 hsa-mir-128b SEQ ID NO: 89 52-73 hsa-mir-129-2 SEQ ID NO: 90 15-35hsa-mir-130a SEQ ID NO: 91 55-74 hsa-mir-130b SEQ ID NO: 92 51-72hsa-mir-132 SEQ ID NO: 93 59-80 hsa-mir-133a-1 SEQ ID NO: 94 54-75hsa-mir-133a-2 SEQ ID NO: 95 60-81 hsa-mir-133b SEQ ID NO: 96 67-87hsa-mir-134 SEQ ID NO: 97 8-28 hsa-mir-135a-1 SEQ ID NO: 98 17-39hsa-mir-135a-2 SEQ ID NO: 99 23-45 hsa-mir-135b SEQ ID NO: 100 16-37hsa-mir-136 SEQ ID NO: 101 15-37 hsa-mir-137 SEQ ID NO: 102 60-81hsa-mir-138-2 SEQ ID NO: 103 10-26 hsa-mir-138-1 SEQ ID NO: 104 23-39hsa-mir-139 SEQ ID NO: 105 7-24 hsa-mir-140 SEQ ID NO: 106 24-44hsa-mir-141 SEQ ID NO: 107 60-80 hsa-mir-142 SEQ ID NO: 108 16-35 and/or52-74 hsa-mir-143 SEQ ID NO: 109 61-82 hsa-mir-144 SEQ ID NO: 110 52-73hsa-mir-145 SEQ ID NO: 111 16-39 hsa-mir-146 SEQ ID NO: 112 21-42hsa-mir-147 SEQ ID NO: 113 47-66 hsa-mir-148a SEQ ID NO: 114 44-65hsa-mir-148b SEQ ID NO: 115 63-84 hsa-mir-149 SEQ ID NO: 116 15-36hsa-mir-150 SEQ ID NO: 117 16-37 hsa-mir-151 SEQ ID NO: 118 46-67hsa-mir-152 SEQ ID NO: 119 54-74 hsa-mir-153-1 SEQ ID NO: 120 54-73hsa-mir-153-2 SEQ ID NO: 121 53-72 hsa-mir-154 SEQ ID NO: 122 15-36hsa-mir-155 SEQ ID NO: 123 4-25 hsa-mir-181a SEQ ID NO: 124 39-61hsa-mir-181b-1 SEQ ID NO: 125 36-59 hsa-mir-181c SEQ ID NO: 126 27-48hsa-mir-181b-2 SEQ ID NO: 127 16-39 hsa-mir-182 SEQ ID NO: 128 23-44and/or 67-87 hsa-mir-183 SEQ ID NO: 129 27-49 hsa-mir-184 SEQ ID NO: 13053-74 hsa-mir-185 SEQ ID NO: 131 15-32 hsa-mir-186 SEQ ID NO: 132 15-37hsa-mir-187 SEQ ID NO: 133 71-91 hsa-mir-188 SEQ ID NO: 134 15-36hsa-mir-190 SEQ ID NO: 135 15-36 hsa-mir-191 SEQ ID NO: 136 16-37hsa-mir-192 SEQ ID NO: 137 24-44 hsa-mir-193 SEQ ID NO: 138 55-75hsa-mir-194-1 SEQ ID NO: 139 15-36 hsa-mir-194-2 SEQ ID NO: 140 15-36hsa-mir-195 SEQ ID NO: 141 15-35 hsa-mir-196-1 SEQ ID NO: 142 7-27hsa-mir-196-2 SEQ ID NO: 143 25-45 hsa-mir-197 SEQ ID NO: 144 48-69hsa-mir-198 SEQ ID NO: 145 6-24 hsa-mir-199a-1 SEQ ID NO: 146 6-28and/or 46-67 hsa-mir-199a-2 SEQ ID NO: 147 31-53 and/or 69-90hsa-mir-199b SEQ ID NO: 148 26-48 hsa-mir-200b SEQ ID NO: 149 54-77hsa-mir-200c SEQ ID NO: 150 45-66 hsa-mir-200a SEQ ID NO: 151 54-75hsa-mir-203 SEQ ID NO: 152 65-86 hsa-mir-204 SEQ ID NO: 153 33-54hsa-mir-205 SEQ ID NO: 154 34-55 hsa-mir-206 SEQ ID NO: 155 53-74hsa-mir-208 SEQ ID NO: 156 44-65 hsa-mir-210 SEQ ID NO: 157 66-86hsa-mir-211 SEQ ID NO: 158 26-47 hsa-mir-212 SEQ ID NO: 159 71-91hsa-mir-213 SEQ ID NO: 160 24-46 and/or 64-85 hsa-mir-214 SEQ ID NO: 16171-91 hsa-mir-215 SEQ ID NO: 162 27-47 hsa-mir-216 SEQ ID NO: 163 19-39hsa-mir-217 SEQ ID NO: 164 35-58 hsa-mir-218-1 SEQ ID NO: 165 25-45hsa-mir-218-2 SEQ ID NO: 166 25-45 hsa-mir-219-1 SEQ ID NO: 167 21-41hsa-mir-219-2 SEQ ID NO: 168 19-39 hsa-mir-220 SEQ ID NO: 169 23-43hsa-mir-221 SEQ ID NO: 170 65-87 hsa-mir-222 SEQ ID NO: 171 69-92hsa-mir-223 SEQ ID NO: 172 68-88 hsa-mir-224 SEQ ID NO: 173 8-30hsa-mir-296 SEQ ID NO: 174 14-34 hsa-mir-299 SEQ ID NO: 175 7-28hsa-mir-301 SEQ ID NO: 176 51-73 hsa-mir-302 SEQ ID NO: 177 44-66hsa-mir-320 SEQ ID NO: 178 48-70 hsa-mir-321 SEQ ID NO: 179 10-30hsa-mir-323 SEQ ID NO: 180 50-71 hsa-mir-324 SEQ ID NO: 181 16-38 and/or51-72 hsa-mir-326 SEQ ID NO: 182 60-79 hsa-mir-328 SEQ ID NO: 183 48-69hsa-mir-330 SEQ ID NO: 184 57-79 hsa-mir-331 SEQ ID NO: 185 61-81hsa-mir-335 SEQ ID NO: 186 16-38 hsa-mir-337 SEQ ID NO: 187 56-78hsa-mir-338 SEQ ID NO: 188 42-64 hsa-mir-339 SEQ ID NO: 189 15-35hsa-mir-340 SEQ ID NO: 190 58-80 hsa-mir-342 SEQ ID NO: 191 61-84hsa-mir-345 SEQ ID NO: 573 17-37 hsa-mir-346 SEQ ID NO: 574 4-26hsa-mir-367 SEQ ID NO: 575 44-65 hsa-mir-368 SEQ ID NO: 576 44-65hsa-mir-369 SEQ ID NO: 577 44-64 hsa-mir-370 SEQ ID NO: 578 48-68hsa-mir-371 SEQ ID NO: 579 44-64 hsa-mir-372 SEQ ID NO: 580 42-64hsa-mir-373 SEQ ID NO: 581 44-66 hsa-mir-374 SEQ ID NO: 582 12-33hsa-mir-375 SEQ ID NO: 677 40-61 hsa-mir-376a SEQ ID NO: 678 44-64hsa-mir-377 SEQ ID NO: 679 45-66 hsa-mir-378 SEQ ID NO: 680 5-26 and44-65 hsa-mir-379 SEQ ID NO: 681 6-24 hsa-mir-380 SEQ ID NO: 682 5-26and 40-61 hsa-mir-381 SEQ ID NO: 683 49-70 hsa-mir-382 SEQ ID NO: 68411-32 hsa-mir-383 SEQ ID NO: 685 7-28 hsa-mir-384 SEQ ID NO: 686 57-76hsa-mir-422a SEQ ID NO: 687 11-32 hsa-mir-423 SEQ ID NO: 688 53-74hsa-mir-424 SEQ ID NO: 689 11-32 hsa-mir-425 SEQ ID NO: 690 55-75hsa-mir-448 SEQ ID NO: 691 71-92 hsa-mir-429 SEQ ID NO: 692 51-72hsa-mir-449 SEQ ID NO: 693 16-37 hsa-mir-450-1 SEQ ID NO: 694 17-38hsa-mir-450-2 SEQ ID NO: 704 22-43 hsa-mir-451 SEQ ID NO: 705 17-39hsa-mir-452 SEQ ID NO: 706 17-38 hsa-mir-453 SEQ ID NO: 707 43-64hsa-mir-455 SEQ ID NO: 708 16-37 hsa-mir-483 SEQ ID NO: 709 48-70hsa-mir-484 SEQ ID NO: 710 2-23 hsa-mir-485 SEQ ID NO: 711 9-30hsa-mir-486 SEQ ID NO: 712 4-25 hsa-mir-487 SEQ ID NO: 713 49-70hsa-mir-488 SEQ ID NO: 714 14-34 hsa-mir-489 SEQ ID NO: 715 51-73hsa-mir-490 SEQ ID NO: 716 76-97 hsa-mir-491 SEQ ID NO: 717 16-38hsa-mir-492 SEQ ID NO: 718 30-52 hsa-mir-493 SEQ ID NO: 719 16-37hsa-mir-494 SEQ ID NO: 720 48-71 hsa-mir-495 SEQ ID NO: 721 50-72hsa-mir-496 SEQ ID NO: 722 61-77 hsa-mir-497 SEQ ID NO: 723 24-44hsa-mir-498 SEQ ID NO: 724 34-56 hsa-mir-499 SEQ ID NO: 725 33-55hsa-mir-500 SEQ ID NO: 726 52-73 hsa-mir-501 SEQ ID NO: 727 14-35hsa-mir-502 SEQ ID NO: 728 1-21 hsa-mir-503 SEQ ID NO: 729 6-28hsa-mir-504 SEQ ID NO: 730 13-33 hsa-mir-505 SEQ ID NO: 731 52-73hsa-mir-506 SEQ ID NO: 732 71-91 hsa-mir-507 SEQ ID NO: 733 56-76hsa-mir-508 SEQ ID NO: 734 61-83 hsa-mir-509 SEQ ID NO: 735 55-77hsa-mir-510 SEQ ID NO: 736 10-32 hsa-mir-511-1 SEQ ID NO: 737 16-36hsa-mir-511-2 SEQ ID NO: 738 16-36 hsa-mir-512-1 SEQ ID NO: 739 14-36hsa-mir-512-2 SEQ ID NO: 740 20-42 hsa-mir-513-1 SEQ ID NO: 741 37-58hsa-mir-513-2 SEQ ID NO: 742 36-57 hsa-mir-514-1 SEQ ID NO: 743 39-58hsa-mir-514-2 SEQ ID NO: 744 39-58 hsa-mir-514-3 SEQ ID NO: 745 39-58hsa-mir-515-1 SEQ ID NO: 746 14-37 hsa-mir-515-2 SEQ ID NO: 747 14-37hsa-mir-516-1 SEQ ID NO: 748 61-78 hsa-mir-516-2 SEQ ID NO: 749 61-78hsa-mir-516-3 SEQ ID NO: 750 15-37 hsa-mir-516-4 SEQ ID NO: 751 15-37hsa-mir-517a SEQ ID NO: 752 15-36 hsa-mir-517b SEQ ID NO: 753 6-27hsa-mir-517c SEQ ID NO: 754 20-41 hsa-mir-518a-1 SEQ ID NO: 755 14-34hsa-mir-518a-2 SEQ ID NO: 756 15-34 hsa-mir-518b SEQ ID NO: 757 51-72hsa-mir-518c SEQ ID NO: 758 24-46 hsa-mir-518d SEQ ID NO: 759 16-36hsa-mir-518e SEQ ID NO: 760 54-75 hsa-mir-518f SEQ ID NO: 761 16-38hsa-mir-519a-1 SEQ ID NO: 762 15-38 hsa-mir-519a-2 SEQ ID NO: 763 54-78hsa-mir-519b SEQ ID NO: 764 13-36 hsa-mir-519c SEQ ID NO: 765 16-39hsa-mir-519d SEQ ID NO: 766 54-76 hsa-mir-519e SEQ ID NO: 767 14-35hsa-mir-520a SEQ ID NO: 768 15-35 hsa-mir-520b SEQ ID NO: 769 41-61hsa-mir-520c SEQ ID NO: 770 16-36 hsa-mir-520d SEQ ID NO: 771 15-37hsa-mir-520e SEQ ID NO: 772 54-74 hsa-mir-520f SEQ ID NO: 773 55-76hsa-mir-520g SEQ ID NO: 774 55-78 hsa-mir-520h SEQ ID NO: 775 55-76hsa-mir-521-1 SEQ ID NO: 776 54-75 hsa-mir-521-2 SEQ ID NO: 777 54-75hsa-mir-522 SEQ ID NO: 778 16-39 hsa-mir-523 SEQ ID NO: 779 16-39hsa-mir-524 SEQ ID NO: 780 16-37 hsa-mir-525 SEQ ID NO: 781 15-35hsa-mir-526a-1 SEQ ID NO: 782 15-35 hsa-mir-526a-2 SEQ ID NO: 783 7-27hsa-mir-526b SEQ ID NO: 784 14-37 hsa-mir-527 SEQ ID NO: 785 14-34ambi-mir-7100 SEQ ID NO: 803 mir-526b* SEQ ID NO: 804 mir-520a* SEQ IDNO: 805

TABLE 2 Mouse miRNA Sequences Processed Sequence miRNA name PrecursorRelative to Precursor mmu-mir-1-1 SEQ ID NO: 192 49-69 mmu-mir-1-2 SEQID NO: 193 47-67 mmu-let-7g SEQ ID NO: 194 7-27 mmu-let-7i SEQ ID NO:195 6-24 mmu-let-7d SEQ ID NO: 196 16-36 + 70-91 mmu-let-7a-1 SEQ ID NO:197 13-34 mmu-let-7a-2 SEQ ID NO: 198 17-38 mmu-let-7b SEQ ID NO: 1997-28 mmu-let-7c-1 SEQ ID NO: 200 16-37 mmu-let-7c-2 SEQ ID NO: 201 14-35mmu-let-7e SEQ ID NO: 202 15-35 mmu-let-7f-1 SEQ ID NO: 203 8-29mmu-let-7f-2 SEQ ID NO: 204 8-29 mmu-mir-7-1 SEQ ID NO: 205 24-44mmu-mir-7-2 SEQ ID NO: 206 19-39 mmu-mir-7b SEQ ID NO: 207 30-50mmu-mir-9-2 SEQ ID NO: 208 8-30 and/or 46-66 mmu-mir-9-1 SEQ ID NO: 20916-38 and/or 56-76 mmu-mir-9-3 SEQ ID NO: 210 16-38 and/or 56-76mmu-mir-10b SEQ ID NO: 211 7-28 mmu-mir-10a-1 SEQ ID NO: 212 22-44mmu-mir-10a-2 SEQ ID NO: 213 22-44 mmu-mir-15b SEQ ID NO: 214 4-25mmu-mir-15a SEQ ID NO: 215 15-36 mmu-mir-16-1 SEQ ID NO: 216 16-37mmu-mir-16-2 SEQ ID NO: 217 17-38 mmu-mir-17 SEQ ID NO: 218 14-37 and/or51-70 mmu-mir-18 SEQ ID NO: 219 17-38 mmu-mir-19b-2 SEQ ID NO: 220 54-76mmu-mir-19a SEQ ID NO: 221 49-71 mmu-mir-19b-1 SEQ ID NO: 222 54-76mmu-mir-20 SEQ ID NO: 223 27-49 mmu-mir-21 SEQ ID NO: 224 18-39mmu-mir-22 SEQ ID NO: 225 57-78 mmu-mir-23b SEQ ID NO: 226 46-68mmu-mir-23a SEQ ID NO: 227 46-66 mmu-mir-24-1 SEQ ID NO: 228 6-28 and/or44-65 mmu-mir-24-2 SEQ ID NO: 229 61-82 mmu-mir-25 SEQ ID NO: 230 52-73mmu-mir-26a-1 SEQ ID NO: 231 16-37 mmu-mir-26b SEQ ID NO: 232 15-36mmu-mir-26a-2 SEQ ID NO: 233 14-35 mmu-mir-27b SEQ ID NO: 234 49-68mmu-mir-27a SEQ ID NO: 235 56-76 mmu-mir-28 SEQ ID NO: 236 14-35mmu-mir-29b-1 SEQ ID NO: 237 47-68 mmu-mir-29a SEQ ID NO: 238 53-74mmu-mir-29c SEQ ID NO: 239 54-75 mmu-mir-29b-2 SEQ ID NO: 240 52-73mmu-mir-30a SEQ ID NO: 241 47-68 mmu-mir-30b SEQ ID NO: 242 2-22mmu-mir-30e SEQ ID NO: 243 2-21 mmu-mir-30c-1 SEQ ID NO: 244 17-39mmu-mir-30c-2 SEQ ID NO: 245 14-36 mmu-mir-30d SEQ ID NO: 246 12-33mmu-mir-31 SEQ ID NO: 247 28-49 mmu-mir-32 SEQ ID NO: 248 6-26mmu-mir-33 SEQ ID NO: 249 6-24 mmu-mir-34c SEQ ID NO: 250 13-35mmu-mir-34b SEQ ID NO: 251 13-35 mmu-mir-34a SEQ ID NO: 252 20-42mmu-mir-92-2 SEQ ID NO: 253 55-75 mmu-mir-92-1 SEQ ID NO: 254 50-70mmu-mir-93 SEQ ID NO: 255 15-37 mmu-mir-96 SEQ ID NO: 256 24-46mmu-mir-98 SEQ ID NO: 257 2-23 mmu-mir-99a SEQ ID NO: 258 6-25mmu-mir-99b SEQ ID NO: 259 7-28 mmu-mir-100 SEQ ID NO: 260 13-34mmu-mir-101 SEQ ID NO: 261 38-57 mmu-mir-101b SEQ ID NO: 262 61-82mmu-mir-103-1 SEQ ID NO: 263 52-74 mmu-mir-103-2 SEQ ID NO: 264 52-74mmu-mir-106a SEQ ID NO: 265 5-26 mmu-mir-106b SEQ ID NO: 266 12-32mmu-mir-107 SEQ ID NO: 267 52-74 mmu-mir-122a SEQ ID NO: 268 6-28mmu-mir-124a-3 SEQ ID NO: 269 43-64 mmu-mir-124a-1 SEQ ID NO: 270 52-73mmu-mir-124a-2 SEQ ID NO: 271 61-82 mmu-mir-125a SEQ ID NO: 272 6-28mmu-mir-125b-2 SEQ ID NO: 273 7-28 mmu-mir-125b-1 SEQ ID NO: 274 15-36mmu-mir-126 SEQ ID NO: 275 9-29 and/or 46-66 mmu-mir-127 SEQ ID NO: 27643-64 mmu-mir-128a SEQ ID NO: 277 44-65 mmu-mir-128b SEQ ID NO: 27848-69 mmu-mir-129-1 SEQ ID NO: 279 6-27 mmu-mir-129-2 SEQ ID NO: 28015-36 mmu-mir-130a SEQ ID NO: 281 42-61 mmu-mir-130b SEQ ID NO: 28251-72 mmu-mir-132 SEQ ID NO: 283 42-63 mmu-mir-133a-1 SEQ ID NO: 28444-65 mmu-mir-133a-2 SEQ ID NO: 285 60-81 mmu-mir-133b SEQ ID NO: 28667-87 mmu-mir-134 SEQ ID NO: 287 7-27 mmu-mir-135a-1 SEQ ID NO: 28817-39 mmu-mir-135b SEQ ID NO: 289 16-37 mmu-mir-135a-2 SEQ ID NO: 29023-45 mmu-mir-136 SEQ ID NO: 291 5-27 mmu-mir-137 SEQ ID NO: 292 46-67mmu-mir-138-2 SEQ ID NO: 293 2-18 mmu-mir-138-1 SEQ ID NO: 294 23-39mmu-mir-139 SEQ ID NO: 295 7-24 mmu-mir-140 SEQ ID NO: 296 7-27mmu-mir-141 SEQ ID NO: 297 49-69 mmu-mir-142 SEQ ID NO: 298 4-23 and/or40-61 mmu-mir-143 SEQ ID NO: 299 40-61 mmu-mir-144 SEQ ID NO: 300 43-64mmu-mir-145 SEQ ID NO: 301 7-30 mmu-mir-146 SEQ ID NO: 302 6-27mmu-mir-148a SEQ ID NO: 303 61-82 mmu-mir-149 SEQ ID NO: 304 4-25mmu-mir-150 SEQ ID NO: 305 6-27 mmu-mir-151 SEQ ID NO: 306 43-63mmu-mir-152 SEQ ID NO: 307 47-67 mmu-mir-153 SEQ ID NO: 308 44-63mmu-mir-154 SEQ ID NO: 309 6-27 mmu-mir-155 SEQ ID NO: 310 4-25mmu-mir-181a SEQ ID NO: 311 7-29 mmu-mir-181b-1 SEQ ID NO: 312 12-35mmu-mir-181c SEQ ID NO: 313 17-38 mmu-mir-181b-2 SEQ ID NO: 314 16-39mmu-mir-182 SEQ ID NO: 315 7-28 mmu-mir-183 SEQ ID NO: 316 6-28mmu-mir-184 SEQ ID NO: 317 45-66 mmu-mir-185 SEQ ID NO: 318 7-24mmu-mir-186 SEQ ID NO: 319 7-29 mmu-mir-187 SEQ ID NO: 320 40-61mmu-mir-188 SEQ ID NO: 321 6-27 mmu-mir-190 SEQ ID NO: 322 6-27mmu-mir-191 SEQ ID NO: 323 7-28 mmu-mir-192 SEQ ID NO: 324 14-31mmu-mir-193 SEQ ID NO: 325 41-61 mmu-mir-194-1 SEQ ID NO: 326 7-28mmu-mir-194-2 SEQ ID NO: 327 16-37 mmu-mir-195 SEQ ID NO: 328 1-21mmu-mir-196-1 SEQ ID NO: 329 24-44 mmu-mir-196-2 SEQ ID NO: 330 16-36mmu-mir-199a-1 SEQ ID NO: 331 6-28 and/or 45-66 mmu-mir-199a-2 SEQ IDNO: 332 31-53 and/or 69-90 mmu-mir-199b SEQ ID NO: 333 26-48mmu-mir-200b SEQ ID NO: 334 45-67 mmu-mir-200a SEQ ID NO: 335 54-75mmu-mir-200c SEQ ID NO: 336 46-67 mmu-mir-201 SEQ ID NO: 337 6-26mmu-mir-202 SEQ ID NO: 338 45-66 mmu-mir-203 SEQ ID NO: 339 49-69mmu-mir-204 SEQ ID NO: 340 6-28 mmu-mir-205 SEQ ID NO: 341 7-28mmu-mir-206 SEQ ID NO: 342 46-67 mmu-mir-207 SEQ ID NO: 343 52-74mmu-mir-208 SEQ ID NO: 344 50-71 mmu-mir-210 SEQ ID NO: 345 66-86mmu-mir-211 SEQ ID NO: 346 26-47 mmu-mir-212 SEQ ID NO: 347 56-76mmu-mir-213 SEQ ID NO: 348 14-36 and/or 54-75 mmu-mir-214 SEQ ID NO: 34971-91 mmu-mir-215 SEQ ID NO: 350 30-50 mmu-mir-216 SEQ ID NO: 351 7-27mmu-mir-217 SEQ ID NO: 352 34-57 mmu-mir-218-2 SEQ ID NO: 353 25-45mmu-mir-219-1 SEQ ID NO: 354 21-41 mmu-mir-219-2 SEQ ID NO: 355 19-39mmu-mir-221 SEQ ID NO: 356 60-81 mmu-mir-222 SEQ ID NO: 357 49-71mmu-mir-223 SEQ ID NO: 358 68-88 mmu-mir-224 SEQ ID NO: 359 8-30mu-miR-290 SEQ ID NO: 360 15-37 mmu-mir-291 SEQ ID NO: 361 14-35 and/or50-72 mmu-mir-292 SEQ ID NO: 362 12-33 and/or 51-73 mmu-mir-293 SEQ IDNO: 363 48-69 mmu-mir-294 SEQ ID NO: 364 51-72 mmu-mir-295 SEQ ID NO:365 43-65 mmu-mir-296 SEQ ID NO: 366 13-33 mmu-mir-297-1 SEQ ID NO: 36715-35 mmu-mir-297-2 SEQ ID NO: 368 36-56 mmu-mir-298 SEQ ID NO: 36911-32 mmu-mir-299 SEQ ID NO: 370 7-28 mmu-mir-300 SEQ ID NO: 371 51-72mmu-mir-301 SEQ ID NO: 372 51-73 mmu-mir-302 SEQ ID NO: 373 44-66mmu-mir-320 SEQ ID NO: 374 48-70 mmu-mir-321 SEQ ID NO: 375 10-30mmu-mir-323 SEQ ID NO: 376 50-71 mmu-mir-324 SEQ ID NO: 377 18-40 and/or53-74 mmu-mir-325 SEQ ID NO: 378 16-38 mmu-mir-326 SEQ ID NO: 379 60-80mmu-mir-328 SEQ ID NO: 380 61-82 mmu-mir-329 SEQ ID NO: 381 61-82mmu-mir-330 SEQ ID NO: 382 61-83 mmu-mir-331 SEQ ID NO: 383 61-81mmu-mir-337 SEQ ID NO: 384 61-83 mmu-mir-338 SEQ ID NO: 385 61-83mmu-mir-339 SEQ ID NO: 386 16-36 mmu-mir-340 SEQ ID NO: 387 61-83mmu-mir-341 SEQ ID NO: 388 61-81 mmu-mir-342 SEQ ID NO: 389 61-84mmu-mir-344 SEQ ID NO: 390 61-83 mmu-mir-345 SEQ ID NO: 391 16-36mmu-mir-346 SEQ ID NO: 392 16-38 mmu-mir-350 SEQ ID NO: 393 61-84mmu-mir-351 SEQ ID NO: 583 16-39 mmu-mir-370 SEQ ID NO: 584 48-70mmu-mir-376a SEQ ID NO: 585 44-64 mmu-mir-376b SEQ ID NO: 586 51-72mmu-mir-380 SEQ ID NO: 587 40-61 mmu-mir-409 SEQ ID NO: 588 47-69mmu-mir-410 SEQ ID NO: 589 50-71 mmu-mir-411 SEQ ID NO: 590 56-78mmu-mir-412 SEQ ID NO: 591 50-72 mmu-mir-425 SEQ ID NO: 695 54-74mmu-mir-429 SEQ ID NO: 696 51-72 mmu-mir-448 SEQ ID NO: 697 72-93mmu-mir-449 SEQ ID NO: 698 16-37 mmu-mir-450 SEQ ID NO: 699 17-38mmu-mir-451 SEQ ID NO: 786 17-38 mmu-mir-452 SEQ ID NO: 787 17-38mmu-mir-463 SEQ ID NO: 788 4-24 mmu-mir-464 SEQ ID NO: 789 47-69mmu-mir-465 SEQ ID NO: 790 5-27 mmu-mir-466 SEQ ID NO: 791 51-73mmu-mir-467 SEQ ID NO: 792 50-71 mmu-mir-468 SEQ ID NO: 793 53-75mmu-mir-469 SEQ ID NO: 794 6-31 mmu-mir-470 SEQ ID NO: 795 9-29mmu-mir-471 SEQ ID NO: 796 7-29 mmu-mir-483 SEQ ID NO: 797 45-67mmu-mir-484 SEQ ID NO: 798 2-23 mmu-mir-485 SEQ ID NO: 799 9-30mmu-mir-486 SEQ ID NO: 800 4-25

TABLE 3 Rat miRNA Sequences Processed Sequence miRNA name PrecursorRelative to Precursor rno-let-7d SEQ ID NO: 394 14-34 and/or 68-89rno-mir-7-1 SEQ ID NO: 395 19-39 and/or 61-82 rno-let-7a-1 SEQ ID NO:396 13-34 rno-let-7a-2 SEQ ID NO: 397 17-38 rno-let-7b SEQ ID NO: 3987-28 rno-let-7c-1 SEQ ID NO: 399 16-37 rno-let-7c-2 SEQ ID NO: 400 14-35rno-let-7e SEQ ID NO: 401 15-35 rno-let-7f-1 SEQ ID NO: 402 8-29rno-let-7f-2 SEQ ID NO: 403 8-29 rno-let-7i SEQ ID NO: 404 6-24rno-mir-7-2 SEQ ID NO: 405 19-39 rno-mir-7b SEQ ID NO: 406 29-49rno-mir-9-1 SEQ ID NO: 407 16-38 rno-mir-9-3 SEQ ID NO: 408 16-38rno-mir-9-2 SEQ ID NO: 409 16-38 rno-mir-10a SEQ ID NO: 410 22-44rno-mir-10b SEQ ID NO: 411 26-47 rno-mir-15b SEQ ID NO: 412 20-41rno-mir-16 SEQ ID NO: 413 17-38 rno-mir-17 SEQ ID NO: 414 14-37rno-mir-18 SEQ ID NO: 415 17-38 rno-mir-19b-1 SEQ ID NO: 416 54-76rno-mir-19b-2 SEQ ID NO: 417 62-84 rno-mir-19a SEQ ID NO: 418 49-71rno-mir-20 SEQ ID NO: 419 16-38 and/or 52-72 rno-mir-21 SEQ ID NO: 42018-39 rno-mir-22 SEQ ID NO: 421 57-78 rno-mir-23a SEQ ID NO: 422 46-66rno-mir-23b SEQ ID NO: 423 58-80 rno-mir-24-1 SEQ ID NO: 424 44-65rno-mir-24-2 SEQ ID NO: 425 61-82 rno-mir-25 SEQ ID NO: 426 52-73rno-mir-26a SEQ ID NO: 427 16-37 rno-mir-26b SEQ ID NO: 428 15-36rno-mir-27b SEQ ID NO: 429 61-80 rno-mir-27a SEQ ID NO: 430 56-76rno-mir-28 SEQ ID NO: 431 14-35 rno-mir-29b-2 SEQ ID NO: 432 52-73rno-mir-29a SEQ ID NO: 433 53-74 rno-mir-29b-1 SEQ ID NO: 434 51-72rno-mir-29c SEQ ID NO: 435 54-75 rno-mir-30c-1 SEQ ID NO: 436 17-39rno-mir-30e SEQ ID NO: 437 2-21 rno-mir-30b SEQ ID NO: 438 16-36rno-mir-30d SEQ ID NO: 439 12-33 rno-mir-30a SEQ ID NO: 440 47-68rno-mir-30c-2 SEQ ID NO: 441 14-36 rno-mir-31 SEQ ID NO: 442 28-49rno-mir-32 SEQ ID NO: 443 6-26 rno-mir-33 SEQ ID NO: 444 6-24rno-mir-34b SEQ ID NO: 445 13-35 rno-mir-34c SEQ ID NO: 446 13-35rno-mir-34a SEQ ID NO: 447 20-42 rno-mir-92-1 SEQ ID NO: 448 48-68rno-mir-92-2 SEQ ID NO: 449 55-75 rno-mir-93 SEQ ID NO: 450 15-37rno-mir-96 SEQ ID NO: 451 24-46 rno-mir-98 SEQ ID NO: 452 2-23rno-mir-99a SEQ ID NO: 453 13-34 rno-mir-99b SEQ ID NO: 454 7-28rno-mir-100 SEQ ID NO: 455 13-34 rno-mir-101b SEQ ID NO: 456 61-82rno-mir-101 SEQ ID NO: 457 47-68 rno-mir-103-2 SEQ ID NO: 458 52-74rno-mir-103-1 SEQ ID NO: 459 52-74 rno-mir-106b SEQ ID NO: 460 12-32rno-mir-107 SEQ ID NO: 461 52-74 rno-mir-122a SEQ ID NO: 462 15-37rno-mir-124a-3 SEQ ID NO: 463 52-73 rno-mir-124a-1 SEQ ID NO: 464 52-73rno-mir-124a-2 SEQ ID NO: 465 61-82 rno-mir-125a SEQ ID NO: 466 15-37rno-mir-125b-1 SEQ ID NO: 467 15-36 rno-mir-125b-2 SEQ ID NO: 468 17-38rno-mir-126 SEQ ID NO: 469 9-29 and/or 46-66 rno-mir-127 SEQ ID NO: 47057-78 rno-mir-128a SEQ ID NO: 471 50-71 rno-mir-128b SEQ ID NO: 47252-73 rno-mir-129-2 SEQ ID NO: 473 19-40 and/or 61-82 rno-mir-129-1 SEQID NO: 474 6-27 rno-mir-130a SEQ ID NO: 475 55-74 rno-mir-130b SEQ IDNO: 476 51-72 rno-mir-132 SEQ ID NO: 477 59-80 rno-mir-133a SEQ ID NO:478 53-74 rno-mir-134 SEQ ID NO: 479 8-28 rno-mir-135b SEQ ID NO: 48016-37 rno-mir-135a SEQ ID NO: 481 23-45 rno-mir-136 SEQ ID NO: 482 15-37rno-mir-137 SEQ ID NO: 483 60-81 rno-mir-138-2 SEQ ID NO: 484 9-25rno-mir-138-1 SEQ ID NO: 485 23-39 rno-mir-139 SEQ ID NO: 486 7-24rno-mir-140 SEQ ID NO: 487 23-43 and/or 61-84 rno-mir-141 SEQ ID NO: 48859-79 rno-mir-142 SEQ ID NO: 489 16-35 and/or 52-74 rno-mir-143 SEQ IDNO: 490 60-81 rno-mir-144 SEQ ID NO: 491 50-71 rno-mir-145 SEQ ID NO:492 16-39 rno-mir-146 SEQ ID NO: 493 17-38 rno-mir-148b SEQ ID NO: 49461-82 rno-mir-150 SEQ ID NO: 495 16-37 rno-mir-151 SEQ ID NO: 496 16-37and/or 50-71 rno-mir-152 SEQ ID NO: 497 53-73 rno-mir-153 SEQ ID NO: 49853-72 rno-mir-154 SEQ ID NO: 499 15-36 rno-mir-181c SEQ ID NO: 500 24-45rno-mir-181a SEQ ID NO: 501 39-61 rno-mir-181b-1 SEQ ID NO: 502 36-59rno-mir-181b-2 SEQ ID NO: 503 15-38 rno-mir-183 SEQ ID NO: 504 27-49rno-mir-184 SEQ ID NO: 505 47-68 rno-mir-185 SEQ ID NO: 506 14-31rno-mir-186 SEQ ID NO: 507 15-37 rno-mir-187 SEQ ID NO: 508 66-86rno-mir-190 SEQ ID NO: 509 15-36 rno-mir-191 SEQ ID NO: 510 15-36rno-mir-192 SEQ ID NO: 511 24-44 rno-mir-193 SEQ ID NO: 512 54-74rno-mir-194-1 SEQ ID NO: 513 15-36 rno-mir-194-2 SEQ ID NO: 514 15-36rno-mir-195 SEQ ID NO: 515 15-35 rno-mir-196 SEQ ID NO: 516 25-45rno-mir-199a SEQ ID NO: 517 31-53 rno-mir-200c SEQ ID NO: 518 46-67rno-mir-200a SEQ ID NO: 519 54-75 rno-mir-200b SEQ ID NO: 520 54-77rno-mir-203 SEQ ID NO: 521 52-73 rno-mir-204 SEQ ID NO: 522 33-54rno-mir-205 SEQ ID NO: 523 33-54 rno-mir-206 SEQ ID NO: 524 51-72rno-mir-208 SEQ ID NO: 525 50-71 rno-mir-210 SEQ ID NO: 526 66-86rno-mir-211 SEQ ID NO: 527 26-47 rno-mir-212 SEQ ID NO: 528 72-92rno-mir-213 SEQ ID NO: 529 55-76 rno-mir-214 SEQ ID NO: 530 71-91rno-mir-216 SEQ ID NO: 531 19-39 rno-mir-217 SEQ ID NO: 532 32-55rno-mir-218-2 SEQ ID NO: 533 25-45 rno-mir-218-1 SEQ ID NO: 534 25-45rno-mir-219-1 SEQ ID NO: 535 21-41 rno-mir-219-2 SEQ ID NO: 536 19-39rno-mir-221 SEQ ID NO: 537 65-87 rno-mir-222 SEQ ID NO: 538 62-85rno-mir-223 SEQ ID NO: 539 68-88 rno-mir-290 SEQ ID NO: 540 14-36rno-mir-291 SEQ ID NO: 541 14-35 and/or 50-72 rno-mir-292 SEQ ID NO: 54212-33 and/or 51-73 rno-mir-296 SEQ ID NO: 543 13-33 rno-mir-297 SEQ IDNO: 544 26-48 rno-mir-298 SEQ ID NO: 545 11-32 rno-mir-299 SEQ ID NO:546 7-28 rno-mir-300 SEQ ID NO: 547 51-72 rno-mir-301 SEQ ID NO: 54861-85 rno-mir-320 SEQ ID NO: 549 48-70 rno-mir-321 SEQ ID NO: 550 10-30rno-mir-322 SEQ ID NO: 551 61-80 rno-mir-323 SEQ ID NO: 552 50-71rno-mir-324 SEQ ID NO: 553 16-38 and/or 51-72 rno-mir-325 SEQ ID NO: 55416-38 rno-mir-326 SEQ ID NO: 555 60-80 rno-mir-328 SEQ ID NO: 556 48-69rno-mir-329 SEQ ID NO: 557 61-82 rno-mir-330 SEQ ID NO: 558 60-82rno-mir-331 SEQ ID NO: 559 61-81 rno-mir-333 SEQ ID NO: 560 16-35rno-mir-336 SEQ ID NO: 561 16-36 rno-mir-337 SEQ ID NO: 562 60-82rno-mir-338 SEQ ID NO: 563 41-63 rno-mir-339 SEQ ID NO: 564 16-36rno-mir-341 SEQ ID NO: 565 61-81 rno-mir-342 SEQ ID NO: 566 61-84rno-mir-344 SEQ ID NO: 567 61-83 rno-mir-345 SEQ ID NO: 568 16-36rno-mir-346 SEQ ID NO: 569 16-38 rno-mir-349 SEQ ID NO: 570 61-82rno-mir-350 SEQ ID NO: 571 61-84 rno-mir-351 SEQ ID NO: 572 16-39rno-mir-352 SEQ ID NO: 592 61-81 rno-mir-421 SEQ ID NO: 593 10-30rno-mir-429 SEQ ID NO: 700 53-74 rno-mir-448 SEQ ID NO: 701 72-93rno-mir-449 SEQ ID NO: 702 16-37 rno-mir-450 SEQ ID NO: 703 17-38rno-mir-451 SEQ ID NO: 801 17-38 rno-mir-483 SEQ ID NO: 802 45-67

It is understood that an miRNA is derived from genomic sequences or agene. In this respect, the term “gene” is used for simplicity to referto the genomic sequence encoding the precursor miRNA for a given miRNA.However, embodiments of the invention may involve genomic sequences of amiRNA that are involved in its expression, such as a promoter or otherregulatory sequences.

The term “recombinant” may be used and this generally refers to amolecule that has been manipulated in vitro or that is the replicated orexpressed product of such a molecule.

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (one or more strands) ofDNA, RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompass the terms “oligonucleotide” and“polynucleotide,” each as a subgenus of the term “nucleic acid.”

The term “miRNA” generally refers to a single-stranded molecule, but inspecific embodiments, molecules implemented in the invention will alsoencompass a region or an additional strand that is partially (between 10and 50% complementary across length of strand), substantially (greaterthan 50% but less than 100% complementary across length of strand) orfully complementary to another region of the same single-strandedmolecule or to another nucleic acid. Thus, nucleic acids may encompass amolecule that comprises one or more complementary or self-complementarystrand(s) or “complement(s)” of a particular sequence comprising amolecule. For example, precursor miRNA may have a self-complementaryregion, which is up to 100% complementary.

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “anneal” as used herein is synonymous with “hybridize.”The term “hybridization”, “hybridize(s)” or “capable of hybridizing”encompasses the terms “stringent condition(s)” or “high stringency” andthe terms “low stringency” or “low stringency condition(s).”

Synthetic nucleic acids of the invention will comprise, in someembodiments the miRNA sequence of any miRNA described in SEQ IDNOs:1-805, and/or any sequence with the complement thereof. It iscontemplated that nucleic acids sequences of the invention can have,have at least, or have at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150 contiguous nucleotides from SEQ ID NOs:1-805 (or anyranger derivable therein), or be a complement thereof. In otherembodiments, nucleic acids are, are at least, or are at most 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%identical or complementary to the miRNA sequence of SEQ ID NOs:1-805 orto the entire sequence of any of SEQ ID NOs:1-805, or any combination orrange derivable therein.

Moreover, sequences are provided in the appendix. The appendix providesa list of 1) miRNAs that were screened, any one of which can be screenedfor using any array or method of the present invention; 2) the names ofthe probe used to screen for that miRNA; and, 3) the sequence of thenamed probe. It is clear that a particular probe can be used foridentifying the level of expression of one or more target miRNAs, or setof target miRNAs (sets of targeted miRNAs may include completelyunrelated RNAs, in additions to sets that are either related or in thesame gene family). It is contemplated that any of these sequences in theappendix can be used in embodiments of the invention.

1. Nucleobases

As used herein a “nucleobase” refers to a heterocyclic base, such as forexample a naturally occurring nucleobase (i.e., an A, T, G, C or U)found in at least one naturally occurring nucleic acid (i.e., DNA andRNA), and naturally or non-naturally occurring derivative(s) and analogsof such a nucleobase. A nucleobase generally can form one or morehydrogen bonds (“anneal” or “hybridize”) with at least one naturallyoccurring nucleobase in manner that may substitute for naturallyoccurring nucleobase pairing (e.g., the hydrogen bonding between A andT, G and C, and A and U).

“Purine” and/or “pyrimidine” nucleobase(s) encompass naturally occurringpurine and/or pyrimidine nucleobases and also derivative(s) andanalog(s) thereof, including but not limited to, those a purine orpyrimidine substituted by one or more of an alkyl, caboxyalkyl, amino,hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol oralkylthiol moeity. Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.)moieties comprise of from about 1, about 2, about 3, about 4, about 5,to about 6 carbon atoms. Other non-limiting examples of a purine orpyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil,a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, abromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, amethylthioadenine, a N,N-diemethyladenine, an azaadenines, a8-bromoadenine, a 8-hydroxyadenine, a 6-hydroxyaminopurine, a6-thiopurine, a 4-(6-aminohexyl/cytosine), and the like. Other examplesare well known to those of skill in the art.

A nucleobase may be comprised in a nucleoside or nucleotide, using anychemical or natural synthesis method described herein or known to one ofordinary skill in the art. Such nucleobase may be labeled or it may bepart of a molecule that is labeled and contains the nucleobase.

2. Nucleosides

As used herein, a “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (i.e., a “5-carbon sugar”), includingbut not limited to a deoxyribose, a ribose, an arabinose, or aderivative or an analog of a 5-carbon sugar. Non-limiting examples of aderivative or an analog of a 5-carbon sugar include a2′-fluoro-2′-deoxyribose or a carbocyclic sugar where a carbon issubstituted for an oxygen atom in the sugar ring.

Different types of covalent attachment(s) of a nucleobase to anucleobase linker moiety are known in the art. By way of non-limitingexample, a nucleoside comprising a purine (i.e., A or G) or a7-deazapurine nucleobase typically covalently attaches the 9 position ofa purine or a 7-deazapurine to the 1′-position of a 5-carbon sugar. Inanother non-limiting example, a nucleoside comprising a pyrimidinenucleobase (i.e., C, T or U) typically covalently attaches a 1 positionof a pyrimidine to a 1′-position of a 5-carbon sugar (Kornberg andBaker, 1992).

3. Nucleotides

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety”. A backbone moiety generally covalently attaches anucleotide to another molecule comprising a nucleotide, or to anothernucleotide to form a nucleic acid. The “backbone moiety” in naturallyoccurring nucleotides typically comprises a phosphorus moiety, which iscovalently attached to a 5-carbon sugar. The attachment of the backbonemoiety typically occurs at either the 3′- or 5′-position of the 5-carbonsugar. However, other types of attachments are known in the art,particularly when a nucleotide comprises derivatives or analogs of anaturally occurring 5-carbon sugar or phosphorus moiety.

4. Nucleic Acid Analogs

A nucleic acid may comprise, or be composed entirely of, a derivative oranalog of a nucleobase, a nucleobase linker moiety and/or backbonemoiety that may be present in a naturally occurring nucleic acid. RNAwith nucleic acid analogs may also be labeled according to methods ofthe invention. As used herein a “derivative” refers to a chemicallymodified or altered form of a naturally occurring molecule, while theterms “mimic” or “analog” refer to a molecule that may or may notstructurally resemble a naturally occurring molecule or moiety, butpossesses similar functions. As used herein, a “moiety” generally refersto a smaller chemical or molecular component of a larger chemical ormolecular structure. Nucleobase, nucleoside and nucleotide analogs orderivatives are well known in the art, and have been described (see forexample, Scheit, 1980, incorporated herein by reference).

Additional non-limiting examples of nucleosides, nucleotides or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives oranalogs, include those in: U.S. Pat. No. 5,681,947, which describesoligonucleotides comprising purine derivatives that form triple helixeswith and/or prevent expression of dsDNA; U.S. Pat. Nos. 5,652,099 and5,763,167, which describe nucleic acids incorporating fluorescentanalogs of nucleosides found in DNA or RNA, particularly for use asfluorescent nucleic acids probes; U.S. Pat. No. 5,614,617, whichdescribes oligonucleotide analogs with substitutions on pyrimidine ringsthat possess enhanced nuclease stability; U.S. Pat. Nos. 5,670,663,5,872,232 and 5,859,221, which describe oligonucleotide analogs withmodified 5-carbon sugars (i.e., modified 2′-deoxyfuranosyl moieties)used in nucleic acid detection; U.S. Pat. No. 5,446,137, which describesoligonucleotides comprising at least one 5-carbon sugar moietysubstituted at the 4′ position with a substituent other than hydrogenthat can be used in hybridization assays; U.S. Pat. No. 5,886,165, whichdescribes oligonucleotides with both deoxyribonucleotides with 3′-5′internucleotide linkages and ribonucleotides with 2′-5′ internucleotidelinkages; U.S. Pat. No. 5,714,606, which describes a modifiedinternucleotide linkage wherein a 3′-position oxygen of theinternucleotide linkage is replaced by a carbon to enhance the nucleaseresistance of nucleic acids; U.S. Pat. No. 5,672,697, which describesoligonucleotides containing one or more 5′ methylene phosphonateinternucleotide linkages that enhance nuclease resistance; U.S. Pat.Nos. 5,466,786 and 5,792,847, which describe the linkage of asubstituent moiety which may comprise a drug or label to the 2′ carbonof an oligonucleotide to provide enhanced nuclease stability and abilityto deliver drugs or detection moieties; U.S. Pat. No. 5,223,618, whichdescribes oligonucleotide analogs with a 2 or 3 carbon backbone linkageattaching the 4′ position and 3′ position of adjacent 5-carbon sugarmoiety to enhanced cellular uptake, resistance to nucleases andhybridization to target RNA; U.S. Pat. No. 5,470,967, which describesoligonucleotides comprising at least one sulfamate or sulfamideinternucleotide linkage that are useful as nucleic acid hybridizationprobe; U.S. Pat. Nos. 5,378,825, 5,777,092, 5,623,070, 5,610,289 and5,602,240, which describe oligonucleotides with three or four atomlinker moiety replacing phosphodiester backbone moiety used for improvednuclease resistance, cellular uptake and regulating RNA expression; U.S.Pat. No. 5,858,988, which describes hydrophobic carrier agent attachedto the 2′-O position of oligonucleotides to enhanced their membranepermeability and stability; U.S. Pat. No. 5,214,136, which describesoligonucleotides conjugated to anthraquinone at the 5′ terminus thatpossess enhanced hybridization to DNA or RNA; enhanced stability tonucleases; U.S. Pat. No. 5,700,922, which describes PNA-DNA-PNA chimeraswherein the DNA comprises 2′-deoxy-erythro-pentofuranosyl nucleotidesfor enhanced nuclease resistance, binding affinity, and ability toactivate RNase H; and U.S. Pat. No. 5,708,154, which describes RNAlinked to a DNA to form a DNA-RNA hybrid; U.S. Pat. No. 5,728,525, whichdescribes the labeling of nucleoside analogs with a universalfluorescent label.

Additional teachings for nucleoside analogs and nucleic acid analogs areU.S. Pat. No. 5,728,525, which describes nucleoside analogs that areend-labeled; U.S. Pat. Nos. 5,637,683, 6,251,666 (L-nucleotidesubstitutions), and U.S. Pat. No. 5,480,980 (7-deaza-2′deoxyguanosinenucleotides and nucleic acid analogs thereof).

The use of other analogs is specifically contemplated for use in thecontext of the present invention. Such analogs may be used in syntheticnucleic acid molecules of the invention, both throughout the molecule orat selected nucleotides. They include, but are not limited to, 1) ribosemodifications (such as 2° F., 2′ NH₂, 2′N₃, 4′thio, or 2′ O—CH₃) and 2)phosphate modifications (such as those found in phosphorothioates,methyl phosphonates, and phosphoroborates). Such analogs have beencreated to confer stability on RNAs by reducing or eliminating theircapacity to be cleaved by ribonucleases. When these nucleotide analogsare present in RNAs, they can have profoundly positive effects on thestability of the RNAs in animals. It is contemplated that the use ofnucleotide analogs can be used alone or in conjunction with any of thedesign modifications of a synthetic miRNA for any nucleic acid of theinvention.

5. Modified Nucleotides

Both synthetic miRNAs and miRNA inhibitors of the invention specificallycontemplate the use of nucleotides that are modified to enhance theiractivities. Such nucleotides include those that are at the 5′ or 3′terminus of the RNA as well as those that are internal within themolecule. Modified nucleotides used in the complementary strands ofsynthetic miRNAs either block the 5′OH or phosphate of the RNA orintroduce internal sugar modifications that enhance uptake of the activestrand of the synthetic miRNA. Modifications for the miRNA inhibitorsinclude internal sugar modifications that enhance hybridization as wellas stabilize the molecules in cells and terminal modifications thatfurther stabilize the nucleic acids in cells. Further contemplated aremodifications that can be detected by microscopy or other methods toidentify cells that contain the synthetic miRNAs or miRNA inhibitors.

B. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. Though synthetic miRNAs accordingto the invention could be produced using recombinant methods, it ispreferred to produce synthetic miRNAs by chemical synthesis or enzymaticproduction. Likewise, miRNA inhibitors are preferentially produced bychemical synthesis or enzymatic production. Non-synthetic miRNAs can beproduced by a number of methods, including methods involving recombinantDNA technology.

Nucleic acid synthesis is performed according to standard methods. See,for example, Itakura and Riggs (1980). Additionally, U.S. Pat. No.4,704,362, U.S. Pat. No. 5,221,619, and U.S. Pat. No. 5,583,013 eachdescribe various methods of preparing synthetic nucleic acids.Non-limiting examples of a synthetic nucleic acid (e.g., a syntheticoligonucleotide), include a nucleic acid made by in vitro chemicallysynthesis using phosphotriester, phosphite or phosphoramidite chemistryand solid phase techniques such as described in EP 266,032, incorporatedherein by reference, or via deoxynucleoside H-phosphonate intermediatesas described by Froehler et al., 1986 and U.S. Pat. No. 5,705,629, eachincorporated herein by reference. In the methods of the presentinvention, one or more oligonucleotide may be used. Various differentmechanisms of oligonucleotide synthesis have been disclosed in forexample, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which isincorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference.

Oligonucleotide synthesis is well known to those of skill in the art.Various different mechanisms of oligonucleotide synthesis have beendisclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571,5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146,5,602,244, each of which is incorporated herein by reference.

Basically, chemical synthesis can be achieved by the diester method, thetriester method polynucleotides phosphorylase method and by solid-phasechemistry. These methods are discussed in further detail below.

Diester method. The diester method was the first to be developed to ausable state, primarily by Khorana and co-workers. (Khorana, 1979). Thebasic step is the joining of two suitably protected deoxynucleotides toform a dideoxynucleotide containing a phosphodiester bond. The diestermethod is well established and has been used to synthesize DNA molecules(Khorana, 1979).

Triester method. The main difference between the diester and triestermethods is the presence in the latter of an extra protecting group onthe phosphate atoms of the reactants and products (Itakura et al.,1975). The phosphate protecting group is usually a chlorophenyl group,which renders the nucleotides and polynucleotide intermediates solublein organic solvents. Therefore purification's are done in chloroformsolutions. Other improvements in the method include (i) the blockcoupling of trimers and larger oligomers, (ii) the extensive use ofhigh-performance liquid chromatography for the purification of bothintermediate and final products, and (iii) solid-phase synthesis.

Polynucleotide phosphorylase method. This is an enzymatic method of DNAsynthesis that can be used to synthesize many useful oligonucleotides(Gillam et al., 1978; Gillam et al., 1979). Under controlled conditions,polynucleotide phosphorylase adds predominantly a single nucleotide to ashort oligonucleotide. Chromatographic purification allows the desiredsingle adduct to be obtained. At least a trimer is required to start theprocedure, and this primer must be obtained by some other method. Thepolynucleotide phosphorylase method works and has the advantage that theprocedures involved are familiar to most biochemists.

Solid-phase methods. Drawing on the technology developed for thesolid-phase synthesis of polypeptides, it has been possible to attachthe initial nucleotide to solid support material and proceed with thestepwise addition of nucleotides. All mixing and washing steps aresimplified, and the procedure becomes amenable to automation. Thesesyntheses are now routinely carried out using automatic nucleic acidsynthesizers.

Phosphoramidite chemistry (Beaucage and Lyer, 1992) has become by farthe most widely used coupling chemistry for the synthesis ofoligonucleotides. As is well known to those skilled in the art,phosphoramidite synthesis of oligonucleotides involves activation ofnucleoside phosphoramidite monomer precursors by reaction with anactivating agent to form activated intermediates, followed by sequentialaddition of the activated intermediates to the growing oligonucleotidechain (generally anchored at one end to a suitable solid support) toform the oligonucleotide product.

Recombinant methods. Recombinant methods for producing nucleic acids ina cell are well known to those of skill in the art. These include theuse of vectors, plasmids, cosmids, and other vehicles for delivery anucleic acid to a cell, which may be the target cell or simply a hostcell (to produce large quantities of the desired RNA molecule).Alternatively, such vehicles can be used in the context of a cell freesystem so long as the reagents for generating the RNA molecule arepresent. Such methods include those described in Sambrook, 2003,Sambrook, 2001 and Sambrook, 1989, which are hereby incorporated byreference.

In certain embodiments, the present invention concerns nucleic acidmolecules that are not synthetic. In some embodiments, the nucleic acidmolecule has a chemical structure of a naturally occurring nucleic acidand a sequence of a naturally occurring nucleic acid, such as the exactand entire sequence of a single stranded primary miRNA (see Lee 2002), asingle-stranded precursor miRNA, or a single-stranded mature miRNA. Inaddition to the use of recombinant technology, such non-syntheticnucleic acids may be generated chemically, such as by employingtechnology used for creating oligonucleotides.

C. Design of Synthetic miRNAs

Synthetic miRNAs typically comprise two strands, an active strand thatis identical in sequence to the mature miRNA that is being studied and acomplementary strand that is at least partially complementary to theactive strand. The active strand is the biologically relevant moleculeand should be preferentially taken up by the complex in cells thatmodulates translation either through mRNA degradation or translationalcontrol. Preferential uptake of the active strand has two profoundresults: (1) the observed activity of the synthetic miRNA increasesdramatically and (2) non-intended effects induced by uptake andactivation of the complementary strand are essentially eliminated.According to the invention, several synthetic miRNA designs can be usedto ensure the preferential uptake of the active strand.

5′ Blocking Agent. The introduction of a stable moiety other thanphosphate or hydroxyl at the 5′ end of the complementary strand impairsits activity in the miRNA pathway. This ensures that only the activestrand of the synthetic miRNA will be used to regulate translation inthe cell. 5′ modifications include, but are not limited to, NH₂, biotin,an amine group, a lower alkylamine group, an acetyl group, 2′O-Me, DMTO,fluoroscein, a thiol, or acridine or any other group with this type offunctionality.

Other sense strand modifications. The introduction of nucleotidemodifications like 2′-OMe, NH₂, biotin, an amine group, a loweralkylamine group, an acetyl group, DMTO, fluoroscein, a thiol, oracridine or any other group with this type of functionality in thecomplementary strand of the synthetic miRNA can eliminate the activityof the complementary strand and enhance uptake of the active strand ofthe miRNA.

Base mismatches in the sense strand. As with siRNAs (Schwarz 2003), therelative stability of the 5′ and 3′ ends of the active strand of thesynthetic miRNA apparently determines the uptake and activation of theactive by the miRNA pathway. Destabilizing the 5′ end of the activestrand of the synthetic miRNA by the strategic placement of basemismatches in the 3′ end of the complementary strand of the syntheticmiRNA enhances the activity of the active strand and essentiallyeliminates the activity of the complementary strand.

D. Host Cells and Target Cells

The cells used to understand miRNA function may be derived from orcontained in any organism (e.g., plant, animal, protozoan, virus,bacterium, or fungus). The plant may be a monocot, dicot or gynmosperm;the animal may be a vertebrate or invertebrate. Preferred microbes arethose used in agriculture or by industry, and those that a pathogenicfor plants or animals. Fungi include organisms in both the mold andyeast morphologies. Examples of vertebrates include fish and mammals,including cattle, goat, pig, sheep, hamster, mouse, rate and human;invertebrate animals include nematodes, insects, arachnids, and otherarthropods. Preferably, the cell is a vertebrate cell. More preferably,the cell is a mammalian cell.

The cells used to understand miRNA function may be from the germ line orsomatic, totipotent or pluripotent, dividing or non-dividing, parenchymaor epithelium, immortalized or transformed, or the like. The cell can bea gamete or an embryo; if an embryo, it can be a single cell embryo or aconstituent cell or cells from a multicellular embryo. The term “embryo”thus encompasses fetal tissue. The cell used for miRNA functionalanalysis may be an undifferentiated cell, such as a stem cell, or adifferentiated cell, such as from a cell of an organ or tissue,including fetal tissue, or any other cell present in an organism. Celltypes that are differentiated include adipocytes, fibroblasts, myocytes,cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes,lymphocytes, macrophages, neutrophils, eosinophils, basophils, mastcells, leukocytes, granulocytes, keratinocytes, chondrocytes,osteoblasts, osteoclasts, hepatocytes, and cells, of the endocrine orexocrine glands. Alternatively, cells may be qualified as germ cells,nurse cells, epithelial cells, endothelial cells, hormone secretingcells, contractile cells, skeletal muscle cells, cardiac muscle cells,blood cells, or cells from the bone, bone marrow, brain, breast, cervix,colon, gastrointestinal tract, heart, kidney, large intestine, liver,lung, lymph nodes, ovary, pancreas, prostate, small intestine, spine orspinal cord, spleen, stomach, testes, thymus, or uterus.

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations formed by cell division. Itis understood that all progeny may not be identical due to deliberate orinadvertent mutations. A host cell may be “transfected” or“transformed,” which refers to a process by which exogenous nucleic acidis transferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny. As used herein, theterms “engineered” and “recombinant” cells or host cells are intended torefer to a cell into which an exogenous nucleic acid sequence, such as,for example, a small, interfering RNA or a template construct encoding areporter gene has been introduced. Therefore, recombinant cells aredistinguishable from naturally occurring cells that do not contain arecombinantly introduced nucleic acid.

A tissue may comprise a host cell or cells to be transformed orcontacted with a nucleic acid delivery composition and/or an additionalagent. The tissue may be part or separated from an organism. In certainembodiments, a tissue and its constituent cells may comprise, but is notlimited to, blood (e.g., hematopoietic cells (such as humanhematopoietic progenitor cells, human hematopoietic stem cells, CD34⁺cells CD4⁺ cells), lymphocytes and other blood lineage cells), bonemarrow, brain, stem cells, blood vessel, liver, lung, bone, breast,cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial,epithelial, esophagus, facia, fibroblast, follicular, ganglion cells,glial cells, goblet cells, kidney, lymph node, muscle, neuron, ovaries,pancreas, peripheral blood, prostate, skin, skin, small intestine,spleen, stomach, testes.

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, human,primate or murine. In other embodiments the organism may be anyeukaryote or even a prokayrote (e.g., a eubacteria, an archaea), aswould be understood by one of ordinary skill in the art (see, forexample, webpage http://phylogeny.arizona.edu/tree/phylogeny.html). Oneof skill in the art would further understand the conditions under whichto incubate all of the above described host cells to maintain them andto permit their division to form progeny.

E. Labels and Tags

Synthetic miRNAs and miRNA inhibitors may be labeled with a radioactive,enzymatic, colorimetric, or other label or tag for detection orisolation purposes. Nucleic acids may be labeled with fluorescence insome embodiments of the invention. The fluorescent labels contemplatedfor use as conjugates include, but are not limited to, Alexa 350, Alexa430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, FluoresceinIsothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, OregonGreen 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red,Renographin, ROX, SYPRO, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

It is contemplated that synthetic miRNAs and miRNA inhibitors may belabeled with two different labels. Furthermore, fluorescence resonanceenergy transfer (FRET) may be employed in methods of the invention(e.g., Klostermeier et al., 2002; Emptage, 2001; Didenko, 2001, eachincorporated by reference).

A number of techniques for visualizing or detecting labeled nucleicacids are readily available. The reference by Stanley T. Crooke, 2000has a discussion of such techniques (Chapter 6) which is incorporated byreference. Such techniques include, microscopy, arrays, Fluorometry,Light cyclers or other real time PCR™ machines, FACS analysis,scintillation counters, Phosphoimagers, Geiger counters, MRI, CAT,antibody-based detection methods (Westerns, immunofluorescence,immunohistochemistry), histochemical techniques, HPLC (Griffey et al.,1997, spectroscopy, capillary gel electrophoresis (Cummins et al.,1996), spectroscopy; mass spectroscopy; radiological techniques; andmass balance techniques. Alternatively, nucleic acids may be labeled ortagged to allow for their efficient isolation. In other embodiments ofthe invention, nucleic acids are biotinylated.

F. Delivery Methods

The present invention involves in some embodiments delivering a nucleicacid into a cell. This may be done as part of a screening method, or itmay be related to a therapeutic or diagnostic application.

RNA molecules may be encoded by a nucleic acid molecule comprised in avector. The term “vector” is used to refer to a carrier nucleic acidmolecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Vectorsinclude plasmids, cosmids, viruses (bacteriophage, animal viruses, andplant viruses), and artificial chromosomes (e.g., YACs). One of skill inthe art would be well equipped to construct a vector through standardrecombinant techniques, which are described in Sambrook et al., 1989 andAusubel et al., 1996, both incorporated herein by reference. In additionto encoding a modified polypeptide such as modified gelonin, a vectormay encode non-modified polypeptide sequences such as a tag or targetingmolecule. A targeting molecule is one that directs the desired nucleicacid to a particular organ, tissue, cell, or other location in asubject's body.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described

There are a number of ways in which expression vectors may be introducedinto cells. In certain embodiments of the invention, the expressionvector comprises a virus or engineered vector derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kb of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells; they can also be used as vectors. Other viral vectorsmay be employed as expression constructs in the present invention.Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988;Baichwal and Sugden, 1986; Coupar et al., 1988) adeno-associated virus(AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska,1984) and herpesviruses may be employed. They offer several attractivefeatures for various mammalian cells (Friedmann, 1989; Ridgeway, 1988;Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

Other suitable methods for nucleic acid delivery to effect expression ofcompositions of the present invention are believed to include virtuallyany method by which a nucleic acid (e.g., DNA, including viral andnonviral vectors) can be introduced into an organelle, a cell, a tissueor an organism, as described herein or as would be known to one ofordinary skill in the art. Such methods include, but are not limited to,direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE-dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al., 1987); by liposome mediated transfection (Nicolau and Sene,1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment(PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos.5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, andeach incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); or by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985).Through the application of techniques such as these, organelle(s),cell(s), tissue(s) or organism(s) may be stably or transientlytransformed.

II. Screening with Synthetic miRNA and miRNA Inhibitor Libraries

As used in the patent application, screening is a process whereinmultiple miRNA-specific reagents are delivered separately intoindividual cell populations or animals. At one or more designated timesafter delivery, the cell populations or animals are assayed for one ormore phenotypes. Those cells or animals that have a significantlydifferent phenotype than cells or animals in the negative control groupare classified as positives. The miRNA that was being manipulated in thesample is defined as a hit. Hits represent targets for additionalresearch and potential therapeutic development.

In some embodiments, there is a multi-step process for screening. Incertain embodiments, there are four general steps:

(1) Develop Quantitative Assay to Monitor Cellular Process beingStudied.

Assays that measure the intensity of a cellular phenotype range frommicroscopic assays that monitor cell size, cell cycle status, orantibody staining to enzymatic assays that assess the turnover of aspecific substrate in a cell lysate to direct measurements ofbiomolecules or small molecules in lysates, on cells, or in medium.

Critical to the success of a screen is creating an assay that trulymeasures the cellular phenotype and maximizing the signal-to-noise ratioof the assay. Maximizing signal-to-noise involves testing variables likeassay time, assay components, cell type, and length of time betweentransfection and assay. The greater the difference in the assay resultsbetween a positive phenotype and a negative control phenotype, thegreater the spread will be in the screening results and the better theopportunity will be to identify interesting genes.

(2) Optimize Transfection Conditions for the Desired Cells.

The first step in this process is identifying a transfection reagent andplating conditions that maximize the uptake of synthetic miRNAs or miRNAinhibitors while maintaining high cell viability. We find it useful totest 2-5 different transfection reagents when using cell lines or 5-10electroporation conditions when using primary or suspension cells.Transfection can be optimized for the reagent or electroporationcondition that worked best among the conditions tested. ScreeningmiRNA-specific libraries requires conditions for high-throughputtransfection. The inventors have developed and used a rapid process thatfacilitates the transfection of up to 1,000 wells in less than an hourwithout the need for robotics (see delivery below).

(3) Screen

Once the assay and transfection process have been developed, a libraryof synthetic miRNAs or miRNA inhibitors can be introduced sequentiallyinto cells in a 24- or 96-well plate. Triplicate transfections for eachreagent provide enough data for reasonable statistical analysis.

(4) Validate Hits

Validating a hit involves showing that the observed phenotype is due tothe miRNA being targeted. Hits are typically confirmed by delivering adilution series of the miRNA inhibitor or synthetic miRNA thatregistered as a hit into the cell that was originally assayed. It hasbeen the experience of the inventors that true hits show a doseresponse.

A. Synthetic miRNA and miRNA Inhibitor Library Preparation

The present invention concerns the preparation and use of syntheticmiRNA and miRNA inhibitor libraries to induce changes in the activity ofspecific miRNAs in cells. Preparation of synthetic miRNAs and miRNAinhibitors typically involves the chemical synthesis of the active andcomplementary strands of the synthetic miRNA and the single-strandedmiRNA inhibitor using any of the methods described in this application.If the active and complementary strands of the synthetic miRNAs are twodistinct molecules, then the two strands must be hybridized prior todelivery. Hybridization can be achieved by mixing the two nucleic acidstogether in roughly equimolar amounts and incubating for a time and at atemperature that is appropriate for hybridization. The addition of salt(e.g., NaCl or NaOAC) enhances hybridization, as does the inclusion of aheat denaturation step prior to the incubation used for hybridization.

B. Delivery of Synthetic miRNAs and miRNA Inhibitors

Libraries of the invention can be used to sequentially up- ordown-regulate one or more miRNAs in samples. This requires methods forintroducing the synthetic miRNAs and miRNA inhibitors into cell typeswith associated cell assays. Lipid-based transfection is typicallyemployed to introduce the nucleic acids into immortalized cells andelectroporation for primary cells.

Suitable methods for nucleic acid delivery according to the presentinvention are believed to include virtually any method by which anucleic acid (e.g., DNA, RNA, including viral and nonviral vectors) canbe introduced into an organelle, a cell, a tissue or an organism, asdescribed herein or as would be known to one of ordinary skill in theart. Such methods include, but are not limited to, direct delivery ofnucleic acids such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274,5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and5,580,859, each incorporated herein by reference), includingmicroinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215,incorporated herein by reference); by electroporation (U.S. Pat. No.5,384,253, incorporated herein by reference); by calcium phosphateprecipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987;Rippe et al., 1990); by using DEAE-dextran followed by polyethyleneglycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987);by liposome mediated transfection (Nicolau and Sene, 1982; Fraley etal., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989;Kato et al., 1991); by microprojectile bombardment (PCT Application Nos.WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055,5,550,318, 5,538,877 and 5,538,880, and each incorporated herein byreference); by agitation with silicon carbide fibers (Kaeppler et al.,1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated hereinby reference); by Agrobacterium-mediated transformation (U.S. Pat. Nos.5,591,616 and 5,563,055, each incorporated herein by reference); or byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S.Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein byreference); by desiccation/inhibition-mediated DNA uptake (Potrykus etal., 1985). Through the application of techniques such as these,organelle(s), cell(s), tissue(s) or organism(s) may be stably ortransiently transformed.

A variety of compounds have been attached to the ends ofoligonucleotides to facilitate their transport across cell membranes.Short signal peptides found in the HIV TAT, HSV VP22, Drosphilaantennapedia, and other proteins have been found to enable the rapidtransfer of biomolecules across membranes (reviewed by Schwarze 2000).These signal peptides, referred to as Protein Transduction Domains(PTDs), have been attached to oligonucleotides to facilitate theirdelivery into cultured cells. Cholesterols have been conjugated tooligonucleotides to improve their uptake into cells in animals(MacKellar 1992). The terminal cholesterol groups apparently interactwith receptors or lipids on the surfaces of cells and facilitate theinternalization of the modified oligonucleotides. Likewise,poly-1-lysine has been conjugated to oligonucleotides to decrease thenet negative charge and improve uptake into cells (Leonetti 1990).

A variety of compounds have been developed that complex with nucleicacids, deliver them to surfaces of cells, and facilitate their uptake inand release from endosomes. Among these are: (1) a variety of lipidssuch as DOTAP (or other cationic lipid), DDAB, DHDEAB, and DOPE and (2)non-lipid-based polymers like polyethylenimine, polyamidoamine, anddendrimers of these and other polymers. In certain of these embodimentsa combination of lipids is employed such as DOTAP and cholesterol or acholesterol derivative (U.S. Pat. No. 6,770,291, which is herebyincorporated by reference). Several of these reagents have been shown tofacilitate nucleic acid uptake in animals.

The cellular components involved in the miRNA pathway are becomingknown. Proteins that stabilize and/or transport miRNAs within cellsmight enhance the stability and activity of miRNAs because they shouldprotect and guide the bound miRNAs once they are in cells. Mixtures ofmiRNA-transporter proteins and miRNAs could enhance the efficacy ofmiRNA-based therapeutics.

RNAs are hydrophilic molecules by virtue of their anionic phosphate andsugar backbone. Although the nucleobases are hydrophobic, hydrophilicitydominates owing to the extensive hydrogen bonding resulting from thephosphate and sugar residues. The hydrophilic character and anionicbackbone reduces cellular permeation. Conjugation of lipophilic groupslike cholesterol (Manoharan, 2002) and lauric and lithocholic acidderivatives with C32 functionality (Lorenz et al., 2004), have beenshown to improve cellular uptake. Moreover binding of steroid conjugatedoligonucleotides to different lipoproteins in the bloodstream, such asLDL, protect their integrity and govern their biodistribution (Rump etal., 2000). Cholesterol attached to anti-sense molecules (Bijsterboschet al., 2001) and aptamers (Rusconi et al., 2004) has also been shown tostabilize oligonucleotides by allowing binding to lipoproteins.Cholesterol has been demonstrated to enhance uptake and serum stabilityof siRNAs in vitro (Lorenz et al., 2004) and in vivo (Soutschek et al.,2004). Additionally, a number of small molecules like SB-435495 (Blackieet al., (2002), Isradipine (Oravcova et al., 1994), amlodipine (Oravcovaet al., 1994) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (Borlakoglu et al.,1990) could enhance cellular uptake, and improve nuclease resistance bypromoting lipoprotein association.

The present methods and kits may be employed for high volume screening.A library of synthetic miRNAs and/or miRNA inhibitors can be createdusing methods of the invention. This library may then be used in highthroughput assays, including microarrays. Specifically contemplated bythe present inventors are chip-based nucleic acid technologies such asthose described by Ziauddin and Sabatini (2001). Briefly, nucleic acidscan be immobilized on solid supports. Cells can then be overlaid on thesolid support and take up the nucleic acids at the defined locations.The impact on the cells can then be measured to identify cocktails thatare having a desirable effect.

C. Labeling and Labeling Techniques

In some embodiments, the present invention concerns miRNA that arelabeled, such as for screening assays to evaluate the therapeutic ordiagnostic relevance of a particular miRNA species. It is contemplatedthat miRNA may first be isolated (either from a cell in which the miRNAis endogenous to the cell or from a cell in which miRNA is exogenous tothe cell) and/or purified prior to labeling. This may achieve a reactionthat more efficiently labels the miRNA, as opposed to other RNA in asample in which the miRNA is not isolated or purified prior to labeling.In many embodiments of the invention, the label is non-radioactive.Generally, nucleic acids may be labeled by adding labeled nucleotides(one-step process) or adding nucleotides and labeling the addednucleotides (two-step process).

Moreover, miRNA may be labeled as is described in U.S. PatentApplication Ser. No. 60/649,584, which is hereby incorporated byreference. Such nucleotides include those that can be labeled with adye, including a fluorescent dye, or with a molecule such as biotin.Labeled nucleotides are readily available; they can be acquiredcommercially or they can be synthesized by reactions known to those ofskill in the art.

1. Nucleotides for Labeling

Nucleotides for labelling are not naturally occurring nucleotides, butinstead, refer to prepared nucleotides that have a reactive moiety onthem. Specific reactive functionalities of interest include: amino,sulfhydryl, sulfoxyl, aminosulfhydryl, azido, epoxide, isothiocyanate,isocyanate, anhydride, monochlorotriazine, dichlorotriazine, mono- ordihalogen substituted pyridine, mono- or disubstituted diazine,maleimide, epoxide, aziridine, sulfonyl halide, acid halide, alkylhalide, aryl halide, alkylsulfonate, N-hydroxysuccinimide ester, imidoester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyldithio)-propionamide, glyoxal, aldehyde, iodoacetyl, cyanomethyl ester,p-nitrophenyl ester, o-nitrophenyl ester, hydroxypyridine ester,carbonyl imidazole, and the other such chemical groups. In someembodiments, the reactive functionality may be bonded directly to anucleotide, or it may be bonded to the nucleotide through a linkinggroup. The functional moiety and any linker cannot substantially impairthe ability of the nucleotide to be added to the miRNA or to be labeled.Representative linking groups include carbon containing linking groups,typically ranging from about 2 to 18, usually from about 2 to 8 carbonatoms, where the carbon containing linking groups may or may not includeone or more heteroatoms, e.g. S, O, N etc., and may or may not includeone or more sites of unsaturation. Of particular interest in manyembodiments are alkyl linking groups, typically lower alkyl linkinggroups of 1 to 16, usually 1 to 4 carbon atoms, where the linking groupsmay include one or more sites of unsaturation. The functionalizednucleotides (or primers) used in the above methods of functionalizedtarget generation may be fabricated using known protocols or purchasedfrom commercial vendors, e.g., Sigma, Roche, Ambion, and NEN. Functionalgroups may be prepared according to ways known to those of skill in theart, including the representative information found in U.S. Pat. Nos.4,404,289; 4,405,711; 4,337,063 and 5,268,486, and Br. Pat. No.1,529,202, which are all incorporated by reference.

Amine-modified nucleotides are used in several embodiments of theinvention. The amine-modified nucleotide is a nucleotide that has areactive amine group for attachment of the label. It is contemplatedthat any ribonucleotide (G, A, U, or C) or deoxyribonucleotide (G, A, T,or C) can be modified for labeling. Examples include, but are notlimited to, the following modified ribo- and deoxyribo-nucleotides:5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and8-[(6-amino)butyl]-amino-ATP; N⁶-(4-amino)butyl-ATP,N⁶-(6-amino)butyl-ATP, N⁴-[2,2-oxy-bis-(ethylamine)]-CTP;N⁶-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP;5-propargylamino-CTP, 5-propargylamino-UTP; 5-(3-aminoallyl)-dUTP;8-[(4-amino)butyl]-amino-dATP and 8-[(6-amino)butyl]-amino-dATP;N⁶-(4-amino)butyl-dATP, N⁶-(6-amino)butyl-dATP,N⁴-[2,2-oxy-bis-(ethylamine)]-dCTP; N⁶-(6-Amino)hexyl-dATP;8-[(6-Amino)hexyl]-amino-dATP; 5-propargylamino-dCTP, and5-propargylamino-dUTP. Such nucleotides can be prepared according tomethods known to those of skill in the art. Moreover, a person ofordinary skill in the art could prepare other nucleotide entities withthe same amine-modification, such as a 5-(3-aminoallyl)-CTP, GTP, ATP,dCTP, dGTP, dTTP, or dUTP in place of a 5-(3-aminoallyl)-UTP.

2. Labeling Techniques

In some embodiments, nucleic acids are labeled by catalytically addingto the nucleic acid an already labeled nucleotide or nucleotides. One ormore labeled nucleotides can be added to miRNA molecules. See U.S. Pat.No. 6,723,509, which is hereby incorporated by reference.

In other embodiments, an unlabeled nucleotide or nucleotides iscatalytically added to an miRNA, and the unlabeled nucleotide ismodified with a chemical moiety that enables it to be subsequentlylabeled. In embodiments of the invention, the chemical moiety is areactive amine such that the nucleotide is an amine-modified nucleotide.

Examples of amine-modified nucleotides are well known to those of skillin the art, many being commercially available such as from Ambion,Sigma, Jena Bioscience, and TriLink.

In contrast to labeling of cDNA during its synthesis, the issue forlabeling miRNA is how to label the already existing molecule. Thepresent invention concerns the use of an enzyme capable of using a di-or tri-phosphate ribonucleotide or deoxyribonucleotide as a substratefor its addition to an miRNA, a small RNA molecule. Moreover, inspecific embodiments, it involves using a modified di- or tri-phosphateribonucleotide, which is added to the 3′ end of an miRNA. The source ofthe enzyme is not limiting. Examples of sources for the enzymes includeyeast, gram-negative bacteria such as E. coli, lactococcus lactis, andsheep pox virus.

Enzymes capable of adding such nucleotides include, but are not limitedto, poly(A) polymerase, terminal transferase, and polynucleotidephosphorylase. In specific embodiments of the invention, ligase iscontemplated as NOT being the enzyme used to add the label, and instead,a non-ligase enzyme is employed.

Poly(A) polymerase has been cloned from a number of organisms fromplants to humans. It has been shown to catalyze the addition ofhomopolymer tracts to RNA (Martin et al., RNA, 4(2):226-30, 1998).

Terminal transferase catalyzes the addition of nucleotides to the 3′terminus of a nucleic acid.

Polynucleotide phosphorylase can polymerize nucleotide diphosphateswithout the need for a primer.

3. Labels

Labels on miRNA or miRNA probes may be colorimetric (includes visibleand UV spectrum, including fluorescent), luminescent, enzymatic, orpositron emitting (including radioactive). The label may be detecteddirectly or indirectly. Radioactive labels include ¹²⁵I, ³²P, ³³P, and³⁵S. Examples of enzymatic labels include alkaline phosphatase,luciferase, horseradish peroxidase, and β-galactosidase. Labels can alsobe proteins with luminescent properties, e.g., green fluorescent proteinand phicoerythrin.

The colorimetric and fluorescent labels contemplated for use asconjugates include, but are not limited to, Alexa Fluor dyes, BODIPYdyes, such as BODIPY FL; Cascade Blue; Cascade Yellow; coumarin and itsderivatives, such as 7-amino-4-methylcoumarin, aminocoumarin andhydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins anderythrosins; fluorescein and its derivatives, such as fluoresceinisothiocyanate; macrocyclic chelates of lanthanide ions, such as QuantumDye™; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red,tetramethylrhodamine and rhodamine 6G; Texas Red; fluorescent energytransfer dyes, such as thiazole orange-ethidium heterodimer; and, TOTAB.

Specific examples of dyes include, but are not limited to, thoseidentified above and the following: Alexa Fluor 350, Alexa Fluor 405,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514,Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750;amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550,BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and,BODIPY-TR; Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE,Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG,Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA,2′,4′,5′,7′-Tetrabromosulfonefluorescein, and TET.

Specific examples of fluorescently labeled ribonucleotides are availablefrom Molecular Probes, and these include, Alexa Fluor 488-5-UTP,Fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP,Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, andBODIPY TR-14-UTP. Other fluorescent ribonucleotides are available fromAmersham Biosciences, such as Cy3-UTP and Cy5-UTP.

Examples of fluorescently labeled deoxyribonucleotides includeDinitrophenyl (DNP)-11-dUTP, Cascade Blue-7-dUTP, Alexa Fluor488-5-dUTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPYFL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPYTMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, AlexaFluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPYTR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY650/665-14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, Alexa Fluor647-12-OBEA-dCTP.

It is contemplated that nucleic acids may be labeled with two differentlabels. Furthermore, fluorescence resonance energy transfer (FRET) maybe employed in methods of the invention (e.g., Klostermeier et al.,2002; Emptage, 2001; Didenko, 2001, each incorporated by reference).

Alternatively, the label may not be detectable per se, but indirectlydetectable or allowing for the isolation or separation of the targetednucleic acid. For example, the label could be biotin, digoxigenin,polyvalent cations, chelator groups and the other ligands, includeligands for an antibody.

4. Visualization Techniques

A number of techniques for visualizing or detecting labeled nucleicacids are readily available. The reference by Stanley T. Crooke, 2000has a discussion of such techniques (Chapter 6), which is incorporatedby reference. Such techniques include, microscopy, arrays, Fluorometry,Light cyclers or other real time PCR machines, FACS analysis,scintillation counters, Phosphoimagers, Geiger counters, MRI, CAT,antibody-based detection methods (Westerns, immunofluorescence,immunohistochemistry), histochemical techniques, HPLC (Griffey et al.,1997, spectroscopy, capillary gel electrophoresis (Cummins et al.,1996), spectroscopy; mass spectroscopy; radiological techniques; andmass balance techniques.

When two or more differentially colored labels are employed, fluorescentresonance energy transfer (FRET) techniques may be employed tocharacterize the dsRNA. Furthermore, a person of ordinary skill in theart is well aware of ways of visualizing, identifying, andcharacterizing labeled nucleic acids, and accordingly, such protocolsmay be used as part of the invention. Examples of tools that may be usedalso include fluorescent microscopy, a BioAnalyzer, a plate reader,Storm (Molecular Dynamics), Array Scanner, FACS (fluorescent activatedcell sorter), or any instrument that has the ability to excite anddetect a fluorescent molecule.

C. Array Preparation

The present invention can be employed with miRNA arrays, which areordered macroarrays or microarrays of nucleic acid molecules (probes)that are fully or nearly complementary or identical to a plurality ofmiRNA molecules or precursor miRNA molecules and that are positioned ona support material in a spatially separated organization. Macroarraysare typically sheets of nitrocellulose or nylon upon which probes havebeen spotted. Microarrays position the nucleic acid probes more denselysuch that up to 10,000 nucleic acid molecules can be fit into a regiontypically 1 to 4 square centimeters. Microarrays can be fabricated byspotting nucleic acid molecules, e.g., genes, oligonucleotides, etc.,onto substrates or fabricating oligonucleotide sequences in situ on asubstrate. Spotted or fabricated nucleic acid molecules can be appliedin a high density matrix pattern of up to about 30 non-identical nucleicacid molecules per square centimeter or higher, e.g. up to about 100 oreven 1000 per square centimeter. Microarrays typically use coated glassas the solid support, in contrast to the nitrocellulose-based materialof filter arrays. By having an ordered array of miRNA-complementingnucleic acid samples, the position of each sample can be tracked andlinked to the original sample. A variety of different array devices inwhich a plurality of distinct nucleic acid probes are stably associatedwith the surface of a solid support are known to those of skill in theart. Useful substrates for arrays include nylon, glass and silicon Sucharrays may vary in a number of different ways, including average probelength, sequence or types of probes, nature of bond between the probeand the array surface, e.g. covalent or non-covalent, and the like.

Representative methods and apparatus for preparing a microarray havebeen described, for example, in U.S. Pat. Nos. 5,143,854; 5,202,231;5,242,974; 5,288,644; 5,324,633; 5,384,261; 5,405,783; 5,412,087;5,424,186; 5,429,807; 5,432,049; 5,436,327; 5,445,934; 5,468,613;5,470,710; 5,472,672; 5,492,806; 5,525,464; 5,503,980; 5,510,270;5,525,464; 5,527,681; 5,529,756; 5,532,128; 5,545,531; 5,547,839;5,554,501; 5,556,752; 5,561,071; 5,571,639; 5,580,726; 5,580,732;5,593,839; 5,599,695; 5,599,672; 5,610,287; 5,624,711; 5,631,134;5,639,603; 5,654,413; 5,658,734; 5,661,028; 5,665,547; 5,667,972;5,695,940; 5,700,637; 5,744,305; 5,800,992; 5,807,522; 5,830,645;5,837,196; 5,871,928; 5,847,219; 5,876,932; 5,919,626; 6,004,755;6,087,102; 6,368,799; 6,383,749; 6,617,112; 6,638,717; 6,720,138, aswell as WO 93/17126; WO 95/11995; WO 95/21265; WO 95/21944; WO 95/35505;WO 96/31622; WO 97/10365; WO 97/27317; WO 99/35505; WO 09923256; WO09936760; WO0138580; WO 0168255; WO 03020898; WO 03040410; WO 03053586;WO 03087297; WO 03091426; WO03100012; WO 04020085; WO 04027093; EP 373203; EP 785 280; EP 799 897 and UK 8 803 000; the disclosures of whichare all herein incorporated by reference.

It is contemplated that the arrays can be high density arrays, such thatthey contain 100 or more different probes. It is contemplated that theymay contain 1000, 16,000, 65,000, 250,000 or 1,000,000 or more differentprobes. The probes can be directed to targets in one or more differentorganisms. The oligonucleotide probes range from 5 to 50, 5 to 45, 10 to40, or 15 to 40 nucleotides in length in some embodiments. In certainembodiments, the oligonucleotide probes are 20 to 25 nucleotides inlength.

The location and sequence of each different probe sequence in the arrayare generally known. Moreover, the large number of different probes canoccupy a relatively small area providing a high density array having aprobe density of generally greater than about 60, 100, 600, 1000, 5,000,10,000, 40,000, 100,000, or 400,000 different oligonucleotide probes percm². The surface area of the array can be about or less than about 1,1.6, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm².

Moreover, a person of ordinary skill in the art could readily analyzedata generated using an array. Such protocols are disclosed above, andinclude information found in WO 9743450; WO 03023058; WO 03022421; WO03029485; WO 03067217; WO 03066906; WO 03076928; WO 03093810; WO03100448A1, all of which are specifically incorporated by reference.

D. Sample Preparation

It is contemplated that the miRNA of a wide variety of samples can beanalyzed using assays described herein. While endogenous miRNA iscontemplated for use with some embodiments, recombinant miRNA—includingnucleic acids that are complementary or identical to endogenous miRNA orprecursor miRNA—can also be handled and analyzed as described herein.Samples may be biological samples, in which case, they can be fromblood, tissue, organs, semen, saliva, tears, other bodily fluid, hairfollicles, skin, or any sample containing or constituting biologicalcells. Alternatively, the sample may not be a biological sample, but bea chemical mixture, such as a cell-free reaction mixture (which maycontain one or more biological enzymes).

E. Hybridization

After the array is prepared and the miRNA in the sample is labeled, thepopulation of target nucleic acids is contacted with the array underhybridization conditions, where such conditions can be adjusted, asdesired, to provide for an optimum level of specificity in view of theparticular assay being performed. Suitable hybridization conditions arewell known to those of skill in the art and reviewed in Sambrook et al.,1989 and WO 95/21944. Of particular interest in many embodiments is theuse of stringent conditions during hybridization. Stringent conditionsare known to those of skill in the art.

It is specifically contemplated that a single array may be contactedwith multiple samples. The samples may be labeled with different labelsto distinguish the samples. For example, a single array can be contactedwith a tumor tissue sample labeled with Cy3, and normal tissue samplelabeled with Cy5. Differences between the samples for particular miRNAscorresponding to probes on the array can be readily ascertained andquantified.

The small surface area of the array permits uniform hybridizationconditions, such as temperature regulation and salt content. Moreover,because of the small area occupied by the high density arrays,hybridization may be carried out in extremely small fluid volumes (e.g.,about 250 μl or less, including volumes of about or less than about 5,10, 25, 50, 60, 70, 80, 90, 100 μl, or any range derivable therein). Insmall volumes, hybridization may proceed very rapidly.

F. Differential Expression Analyses

Arrays can be used to detect differences between two samples. This canalso be used for diagnostic purposes. Specifically contemplatedapplications include identifying and/or quantifying differences betweenmiRNA from a sample that is normal and from a sample that is not normalor between two differently treated samples. Also, miRNA may be comparedbetween a sample believed to be susceptible to a particular disease orcondition and one believed to be not susceptible or resistant to thatdisease or condition. A sample that is not normal is one exhibitingphenotypic trait(s) of a disease or condition or one believed to be notnormal with respect to that disease or condition. It may be compared toa cell that is normal with respect to that disease or condition.Phenotypic traits include symptoms of, or susceptibility to, a diseaseor condition of which a component is or may or may not be genetic.

G. Cell Assays to Identify miRNAs with Ties to Disease

Specifically contemplated applications include identifying miRNAs thatcontribute to cellular processes that are themselves parts of a diseaseor might otherwise be associated with a particular disease state. Also,miRNA functions may be compared between a sample believed to besusceptible to a particular disease or condition and one believed to benot susceptible or resistant to that disease or condition. It isspecifically contemplated that RNA molecules of the present inventioncan be used to treat any of the diseases or conditions discussed in theprevious section or modulate any of the cellular pathways discussed inthe previous section.

Specifically contemplated applications include identifying miRNAs thatcontribute to cellular processes that are themselves parts of a diseaseor might otherwise be associated with a particular disease state. Also,miRNA functions may be compared between a sample believed to besusceptible to a particular disease or condition and one believed to benot susceptible or resistant to that disease or condition.

AIDS, autoimmune diseases (rheumatoid arthritis, multiple sclerosis,diabetes-insulin-dependent and non-independent, systemic lupuserythematosus and Graves disease); cancer (e.g., malignant, benign,metastatic, precancer); cardiovascular diseases (heart disease orcoronary artery disease, stroke—ischemic and hemorrhagic, and rheumaticheart disease); diseases of the nervous system; and infection bypathogenic microorganisms (Athlete's Foot, Chickenpox, Common cold,Diarrheal diseases, Flu, Genital herpes, Malaria, Meningitis, Pneumonia,Sinusitis, Skin diseases, Strep throat, Tuberculosis, Urinary tractinfections, Vaginal infections, Viral hepatitis); inflammation (allergy,asthma); prion diseases (e.g., CJD, kuru, GSS, FFI).

Moreover, miRNA can be evaluated with respect to the following diseases,conditions, and disorders: Abdominal Adhesions; Anal Abscess; BrainAbscess; Peritonsillar Abscess; Absence Seizures; Achalasia; Acne;Acoustic Neuroma; Acquired Immunodeficiency Syndrome (AIDS);Acrochordon; Actinic Keratosis; Adenocarcinoma of the Lung; ADHD;Adult-Onset Diabetes; Aero-Otitis; Age Spots; Age-Related Hearing Loss;Age-Related Macular Degeneration; Age-Related Vision Change(Presbyopia); Agoraphobia; Alcohol Withdrawal; Alcoholism; AllergenImmunotherapy; Allergic Rhinitis; Allergies; Alopecia (Areata,Hereditary-Patterned, and Traumatic); Altitude Sickness; Alzheimer'sDisease; Amaurotic Familial Infantile Idiocy; Amblyopia; Amenorrhea;Amyloidosis; Amyotrophic Lateral Sclerosis (ALS); Anaphylaxis;Androgenetic Alopecia; Anemia (Aplastic, Hemolytic, Pernicious, andSickle Cell); Angina; Angiomas, Spider; Angioplasty; AnkylosingSpondylitis; Anorexia Nervosa; Anovulatory Bleeding;Antibiotic-Associated Diarrhea; Antiphospholipid Antibody Syndrome;Antisocial Personality Disorder; Anus Fissure, Fistula, Hemorrhoids,Anus Itch, Stricture; Anxiety Disorders (Generalized,Obsessive-Compulsive Disorder, Panic Disorder, Phobia, andPost-Traumatic Stress Disorder); Aortic Aneurysm; Aortic Arch Syndrome;Appendicitis; Arrhythmias, Cardiac; Arteritis, Takayasu's; ArthriticDiseases (Ankylosing Spondylitis, Gout, Infectious, Juvenile,Osteoarthritis, Pseudogout, Psoriatic Arthritis, and Rheumatoid);Asbestosis; Ascending Cholangitis; Asteatotic Eczema; Asthma;Astigmatism; Asymptomatic Bacteriuria; Ataxia, Friedreich's;Atherosclerosis; Athlete's Foot; Atopic Dermatitis; Atrial Fibrillation;Atrophic Vaginitis; Attention-Deficit Hyperactivity Disorder; Autism;Autoimmune Diseases (Celiac Disease, Crohn's Disease, Diabetes Mellitus,Type 1 (Insulin-Dependent; Juvenile-Onset), Diabetes Mellitus, Type 2(Non-Insulin-Dependent; Adult-Onset), Graves' Disease, Hyperthyroidism,Immune Thrombocytopenic Purpura, Lupus, Myasthenia Gravis, PolyarteritisNodosa, Rheumatoid Arthritis, Scleroderma, Takayasu's Arteritis, andUlcerative Colitis); B12 Deficiency; Bacillary Dysentery; BacterialGastroenteritis; Bacterial Vaginosis; Balanitis; Baldness,Hereditary-Patterned; Barber's Itch; Barotitis; Barotrauma; Bartholin'sGland Cyst; Basal-Cell Carcinoma; Bed-Wetting; Bedsores; Behcet'sSyndrome; Bell's Palsy; Bends; Benign Prostatic Hyperplasia; Bile-DuctDiseases; Biliary Colic; Biopsy; Bipolar Disorder; Bladder conditions(Infection; Interstitial Cystitis; Prolapse; Urethritis; UrinaryIncontinence; Urinary Tract Infection); Blepharitis; Blepharoptosis;Blighted Ovum; Friction Blisters; Blood Pressure, High; Boils; Bonediseases and conditions (Osteoporosis; Paget's Disease); Bone Yaws;Borderline Personality Disorder; Bornholm Disease; Botulism; BowelObstruction; Bradycardia; Bronchitis; Bulimia Nervosa; Bunion; Bursitis;C. Difficile Colitis; Calcaneal Apophysitis; Calcium PyrophosphateDeposition Disease; Campylobacteriosis; Cancer; Candidiasis;Carbon-Monoxide Poisoning; Carbuncles; Cardiac Arrhythmias (AtrialFibrillation, Bradycardia); Cardiomyopathy; Carpal Tunnel Syndrome;Cataracts; Cellulitis; Central Serous Retinopathy; Cerebral Palsy;Cerebromacular Degeneration; Cerumen Impaction; Cervicitis, NabothianCysts, Cervical Polyps, Cervical Warts; Chalazion; Chickenpox;Chlamydia; Chloasma; Cholangitis; Cholecystitis; Cholesteatoma;Chondromalacia; Chorea; Choroidal Melanoma; Chronic Bronchitis; ChronicFatigue Syndrome; Chronic Hepatitis; Chronic Leukemia; ChronicObstructive Pulmonary Disease; Chronic Otitis Media; Cirrhosis; ClusterHeadache; Cogan's Syndrome; Cold, Common; Colic, Biliary;Pseudomembranous Colitis, Ulcerative Colitis, Collapsed Lung; CollarboneFracture; Coma; Complex Regional Pain Syndrome; Congestive HeartFailure; Conjunctivitis; Constipation; Contact Dermatitis; ConversionDisorder; COPD; Cornea Abrasion, Cornea Keratitis; Corns; CoronaryArtery Disease; Creutzfeldt-Jakob Disease; Crossed Eyes; Croup;Cryptorchidism; Cystic Fibrosis; Interstitial Cystitis; Cystocele;Cysts; Cytomegalovirus infection; Dacryocystitis; Dandruff;Decompression Sickness; Decubitus Ulcers; Delirium Tremens; DelusionalDisorder; Dementia; Depressive Disorders (Bipolar Disorder, Dysthymia,Major Depression, Manic Depression, Postpartum Depression); Dermatitis;Dermatofibroma; Dermatomyositis; Detached Retina; DevelopmentalDysplasia of the Hip; Deviated Septum; Devil's Grip; Diabetes(Gestational Diabetes; Type 1 Diabetes (Insulin-Dependent; Juvenile);Type 2 Diabetes (Non-Insulin-Dependent; Adult-Onset); Hypoglycemia,Ketoacidosis, Nephropathy, Neuropathies, Retinopathy)Antibiotic-Associated Diarrhea; Diplopia; Herniated Disk; DislocatedLens; Hip Dislocation (Developmental); Diverticulitis; Diverticulosis;Dizziness; Doerderland's Vaginitis; Double Vision; Down Syndrome;Drooping Eyelid; Dry Skin; Sun-Damaged Skin; Dry-Eye Syndrome;Duck-Foot; Dysautonomia, Familial; Dysfunctional Uterine Bleeding;Dyslexia; Dyspareunia; Dysthymia; Dysuria; Eating Disorders (AnorexiaNervosa, Bulimia Nervosa); Eclampsia; Eczema; Edema; Emphysema;Encephalitis; Encopresis; End-Stage Renal Disease; Endocarditis;Endometriosis; Endophthalmitis; Endoscopy; Enlarged Prostate; Enuresis;Epidemic Benign Dry Pleurisy; Epididymitis; Epiglottitis; Epilepsy;Epistaxis; Erectile Dysfunction; Erythema Infectiosum; Esophagitis;Esophagus Achalasia; Esophagitis; Essential Hypertension; EssentialTremor; Ewing's Sarcoma; Familial Dysautonomia; Farsightedness; FebrileSeizures; Fecal Incontinence; Fever; Fever-Induced Seizures; Fibroids;Fibromyalgia; Fifth Disease; Filiform Warts; Flat Warts; Flatulence;Flu; Focal Seizures; Food Allergy; Food Poisoning; Forefoot Neuroma;Fragile X Syndrome; Friction Blisters; Friedreich's Ataxia; Frostbite;Fungal Infections (Athlete's Foot, Brain Abscess, Infectious Arthritis,Jock Itch, Onychomycosis, Ringworm, Swimmer's Ear, Tinea Cruris, TineaUnguium, Tinea Versicolor); Furuncle; Gallstones; Gardnerella Vaginitis;Gastritis; Gastrocnemius Strain; Gastroenteritis; GastroesophagealReflux Disease; Gastrointestinal Amebiasis; Generalized AnxietyDisorder; Generalized Barotrauma; Genital Herpes; Genital Warts; GERD;Germ Cell Tumors, Extragonadal; Giant Cell Arteritis; Giardiasis;Glaucoma; Glomerulonephritis; Gluten-Sensitive Enteropathy; GM2Gangliosidosis; Gonorrhea; Gout; Grand Mal Seizures; Graves' Disease;Graves' Opthalmopathy; Guillain-Barre Syndrome; Hammertoe; Hay Fever;Headache; Hearing Loss; Heart Attack; Heat Stroke; Heel Spur; Heloma;Spider Hemangiomas; Hematoma; Hematuria; Hemochromatosis; HemolyticAnemia; Hemophilia; Hemorrhagic Stroke; Subarachnoid Hemorrhagic Stroke;Hemorrhoids; Hepatitis A; Hepatitis B; Hepatitis C; Hereditary-PatternedBaldness; Hernia; Herniated Disk; High Blood Pressure; High Cholesterol;Hirsutism; Histiocytosis X; HIV/AIDS; Hordeolum; Human Papilloma Virus(HPV); Huntington's Disease; Hydatidiform Mole; Hydrocephalus;Hyperactivity; Hypercholesterolemia; Hyperkeratosis; Hyperopia;Hypertension; Ocular Hypertension; Secondary Hypertension; HypertensiveRetinopathy; Hyperthermia; Hyperthyroidism; Hypochondriasis;Hypoglycemia; Hypoparathyroidism; Hypothyroidism; IBS; ICD; Ichthyosis;Immune Thrombocytopenic Purpura; Impetigo; Impotence; Incontinence;Infantile Ganglioside Lipidosis; Infectious Arthritis; InfectiousMononucleosis; Infertility; Inflammatory Bowel Disease; Inguinal Hernia;Insomnia; Intercerebral Hemorrhage; Interdigital Neuroma;Intermetatarsal Neuroma; Intermittent Claudication; InterstitialCystitis; Intestinal Obstruction; Iron Deficiency; Irritable BowelSyndrome; Juvenile Arthritis; Kaposi's Sarcoma; Kawasaki Syndrome;Keloids; Keratitis; Actinic Keratosis; Labyrinthitis; LactoseIntolerance; Lacunar Stroke; Langerhans' Cell Histiocytosis; Laryngitis;Laryngotracheitis; Lateral Epicondylitis; Latex Allergy; Lazy Eye; LeadPoisoning; Intermittent Claudication; Restless Legs Syndrome; ShinSplints; Leg Strain; Cataract; Dislocated Lens; Leukemia; Lice; LichenSimplex Chronicus; Cirrhosis; Hepatitis; Liver Spots; Lockjaw; LouGehrig's Disease; Lupus Erythematosus, Systemic; Lyme Disease;Lymphedema; Lymphoma; Macular Degeneration; Malabsorption Syndromes;Malaria; Male Pattern Baldness; Malignant Hyperthermia; ManicDepression; Marfan's Syndrome; Mastoiditis; Measles; Meckel'sDiverticulum; Melasma; Meniere's Disease; Meningitis; Menopause; MentalRetardation; Phenylketonuria; Migraine; Miscarriage; Mitral-ValveProlapse; Mittelschmerz; Molar Pregnancy; Molluscum Contagiosum;Mononucleosis; Morton's Neuroma; Mosaic Warts; Motor Tics; MucocutaneousLymph Node Syndrome; Multiple Sclerosis; Mumps; Muscular Dystrophy;Musculoskeletal Disorders (Fibromyalgia, Giant Cell Arteritis, Gout,Infectious Arthritis, Muscular Dystrophy, Myositis, Osteoarthritis,Osteoporosis, Paget's Disease Of Bone, Polymyalgia Rheumatica,Pseudogout, Reflex Sympathetic Dystrophy, Rheumatoid Arthritis,Scleroderma, Systemic Lupus Erythematosus, Tendonitis); MyastheniaGravis; Myocardial Infarction; Myocarditis; Myopia; Myositis; NailFelon; Onycholysis; Onychomycosis; Paronychia; Subungual Hematoma;Narcolepsy; Nasal Polyps; Nausea; Nearsightedness; Needle Biopsy;Nephrectomy; Nephroblastoma; Nephrolithiasis; Nephropathy, Diabetic;Neuritis, Retrobulbar; Neuroblastoma; Neuromuscular Disorders;Neuropathies; Guillain-Barre Syndrome; Retrobulbar; Nevi; NevusFlammeus; Nevus Simplex; Nocturnal Enuresis; Non-Tropical Sprue;Obesity; Obsessive-Compulsive Disorder; Occupational Hearing Loss;Ocular Hypertension; Ocular Rosacea; Onycholysis; Onychomycosis;Glaucoma; Retrobulbar Neuritis; Optic Nerve Swelling; Orbit Fracture;Orchitis; Osgood-Schlatter Disease; Osteoarthritis; Osteoporosis;Osteosarcoma; Otitis Externa; Otitis Media; Chronic Otitis Media;Otosclerosis; Ototoxicity; Pelvic Inflammatory Disease; Polycystic OvarySyndrome; Painful-Bladder Syndrome; Pancreatitis; Panic Disorder;Papilledema; Paraphimosis; Parkinson's Disease; Paronychia; PartialSeizures; PCL Injuries; Pedunculated Warts; Pelvic Relaxation;Paraphimosis; Peyronie's Disease; Peptic Ulcer; Perforated Eardrum;Pericarditis; Perimenopause; Peripheral Vascular Disease; PeritonsillarAbscess; Persistent Vegetative State; Personality Disorders; Petit MalSeizures; Peyronie's Disease; Pharyngitis; Pharynx Cancer;Phenylketonuria; Phimosis; Phobia; Photosensitivity; PigmentationDisorders (Chloasma, Melasma, Vitiligo); Piles; Pinkeye; PityriasisRosea; PKU; Plague; Plantar Fasciitis; Plantar Warts; Plantaris Strain;Pleurisy; Pleurodynia; PMS; Pneumoconiosis; Pneumonectomy; Pneumonia;Pneumothorax; Lead Poisoning; Polio; Poliomyelitis; PolyarteritisNodosa; Polychondritis; Polymyalgia Rheumatica; Polymyositis; ColonicPolyps; Nasal Polyps; Vocal Cord Polyps; Port-Wine Stain; Post-PolioSyndrome; Postinfectious Thrombocytopenia; Postpartum Depression;Preeclampsia; Pregnancy-Induced Hypertension; Premenstrual Syndrome;Pressure Sores; Primary Sclerosing Cholangitis; Prolapse; EnlargedProstate; Acute Prostatitis; Chronic Prostatitis; Pruritus Ani;Pseudogout; Psoriasis; Psoriatic Arthritis; Ptosis; Pulseless Disease;Pyelonephritis; Quadriceps Strain; Quinsy; Rash; Raynaud's Phenomenon;Rectal Itch; Rectocele; Reflex Sympathetic Dystrophy; Renal Failure;Respiratory Disorders Respiratory Syncytial Virus; Retina Detachment;Retinitis Pigmentosa; Retinopathy; Retrobulbar Neuritis; Reye'sSyndrome; Rhabdomyosarcoma; Rheumatoid Arthritis; Allergic Rhinitis;Viral Rhinitis (Common Cold); Riley-Day Syndrome; Ringworm; RockyMountain Spotted Fever; Rosacea; Rubeola; Mumps; Salivary GlandDisorders; Salmon Patch; Sarcoidosis; Scabies; Scarlet Fever; Scars;Schizophrenia; Schizotypal Personality Disorder; Sciatica; Scleritis;Scleroderma; Scoliosis; Sebaceous Cysts; Seborrhea; SeborrheicKeratoses; Secondary Hypertension; Seizures; Sexual Dysfunction;Sexually Transmitted Diseases; Shigellosis; Shingles; Sialadenitis;Sialadenosis; Sialolithiasis; Sickle-Cell Anemia; Siderosis; Silicosis;Sinus Cancer; Sjögren's Syndrome; Sleep Disorders; Smallpox; SocialAnxiety Disorder; Solar Lentigo; Somatoform Disorders (Hypochondriasis,Somatization Disorder); Somnambulism; Spastic Colon; Spider Veins; SpinaBifida; Spinal Cord Trauma; Spontaneous Abortion; Stasis Dermatitis;Strabismus; Strep Throat; Streptococcal Toxic Shock Syndrome; Stroke;Subarachnoid Hemorrhage; Transient Ischemic Attack; Stuttering;Subungual Hematoma; Sun Allergy; Sun-Damaged Skin; Sylvest's Disease;Systemic Lupus Erythematosus; Systemic Sclerosis; Tachycardia;Takayasu's Arteritis; Tay-Sachs Disease; Tear-Duct Infection; TelogenEffluvium; Temporal Arteritis; Tendonitis; Tennis Elbow; TensionHeadache; Testicular Torsion; Undescended Testicles; Tetanus;Thrombocytopenia; Thrombophlebitis; Thrombotic Stroke; Tinea; Tinnitus;Tonsillitis; Torsional Deformities; Toxemia Of Pregnancy; Toxic ShockSyndrome, Streptococcal; Toxoplasmosis; Trichomoniasis; TrigeminalNeuralgia (Tic Douloureux); Tuberculosis; Tylosis; Ulcer; Urethritis;Urinary Tract disorders and conditions; Uroliniasis; Urticaria; Uterinedisorders; Uterine Prolapse; Uveitis; Vaginitis; Bacterial (Gardnerella)Vaginosis; Varicella; Varices, Esophageal; Varicose Veins; VascularDisorders (Hypertension, Intermittent Claudication, Peripheral VascularDisease, Polyarteritis Nodosa, Raynaud's Phenomenon, Takayasu'sArteritis, Thrombophlebitis, Vasculitis, Wegener's Granulomatosis); VeinInflammation; Varicose Veins; Vertigo; Vestibular Schwannoma; ViralRhinitis; Vitamin B12 Deficiency; Vitiligo; Vocal Tics; Vocal-CordDisorders; Common Warts; Genital Warts; Plantar Warts; Water On TheBrain; Wax Blockage Of Ear Canal; Esophageal Webs; Werlhofs Disease;Wrinkles; Yersinia Pestis Infection. It is contemplated that suchdiseases can be diagnosed or treated using a nucleic acids of theinvention that correspond to miRNAs.

Cancers that may be evaluated, diagnosed, and/or treated by methods andcompositions of the invention include cancer cells from the bladder,blood, bone, bone marrow, brain, breast, colon, esophagus,gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck,ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition,the cancer may specifically be of the following histological type,though it is not limited to these: neoplasm, malignant; carcinoma;carcinoma, undifferentiated; giant and spindle cell carcinoma; smallcell carcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; maligmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia. Moreover, miRNA can be evaluated in precancers, such asmetaplasia, dysplasia, and hyperplasia.

It is specifically contemplated that the invention can be used toevaluate or diagnose differences between stages of disease, such asbetween pre-cancer and cancer, or between a primary tumor and ametastasized tumor.

The efficacy of different therapeutic drugs is altered by miRNAsaccording to the present invention. Such therapeutic drugs include, butare not limited to, chemotherapeutic drugs. A “chemotherapeutic agent”is used to connote a compound or composition that is administered in thetreatment of cancer. These agents or drugs are categorized by their modeof activity within a cell, for example, whether and at what stage theyaffect the cell cycle. Alternatively, an agent may be characterizedbased on its ability to directly cross-link DNA, to intercalate intoDNA, or to induce chromosomal and mitotic aberrations by affectingnucleic acid synthesis. Most chemotherapeutic agents fall into thefollowing categories: alkylating agents, antimetabolites, antitumorantibiotics, mitotic inhibitors, and nitrosoureas.

Examples of chemotherapeutic agents include alkylating agents such asthiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammalI and calicheamicinomegal1; dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumcoordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11);topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY17018, onapristone, and toremifene; aromatase inhibitorsthat inhibit the enzyme aromatase, which regulates estrogen productionin the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, megestrol acetate, exemestane, formestanie,fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens suchas flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; aswell as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);antisense oligonucleotides, particularly those which inhibit expressionof genes in signaling pathways implicated in aberrant cellproliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF expression inhibitor and a HER2 expressioninhibitor; vaccines such as gene therapy vaccines and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. A list ofU.S. FDA approved oncology drugs with their approved indications can befound on the World Wide Web ataccessdata.fda.gov/scripts/cder/onctools/druglist.cfm. Moreover, it iscontemplated that samples that have differences in the activity ofcertain pathways may also be compared. Such cellular pathways includebut are not limited to the following: any adhesion or motility pathwayincluding but not limited to those involving cyclic AMP, protein kinaseA, G-protein couple receptors, adenylyl cyclase, L-selectin, E-selectin,PECAM, VCAM-1, α-actinin, paxillin, cadherins, AKT, integrin-α,integrin-β, RAF-1, ERK, PI-3 kinase, vinculin, matrixmetalloproteinases, Rho GTPases, p85, trefoil factors, profilin, FAK,MAP kinase, Ras, caveolin, calpain-1, calpain-2, epidermal growth factorreceptor, ICAM-1, ICAM-2, cofilin, actin, gelsolin, RhoA, RAC1, myosinlight chain kinase, platelet-derived growth factor receptor or ezrin;any apoptosis pathway including but not limited to those involving AKT,Fas ligand, NF_(K)B, caspase-9, PI3 kinase, caspase-3, caspase-7, ICAD,CAD, EndoG, Granzyme B, Bad, Bax, Bid, Bak, APAF-1, cytochrome C, p53,ATM, Bcl-2, PARP, Chk1, Chk2, p21, c-Jun, p73, Rad51, Mdm2, Rad50,c-Abl, BRCA-1, perforin, caspase-4, caspase-8, caspase-6, caspase-1,caspase-2, caspase-10, Rho, Jun kinase, Jun kinase kinase, Rip2,lamin-A, lamin-B1, lamin-B2, Fas receptor, H₂O₂, Granzyme A, NADPHoxidase, HMG2, CD4, CD28, CD3, TRADD, IKK, FADD, GADD45, DR3 deathreceptor, DR4/5 death receptor, FLIPs, APO-3, GRB2, SHC, ERK, MEK,RAF-1, cyclic AMP, protein kinase A, E2F, retinoblastoma protein,Smac/Diablo, ACH receptor, 14-3-3, FAK, SODD, TNF receptor, RIP,cyclin-D1, PCNA, Bcl-XL, PIP2, PIP3, PTEN, ATM, Cdc2, protein kinase C,calcineurin, IKKα, IKKβ, IKKγ, SOS-1, c-FOS, Traf-1, Traf-2, I_(K)Bβ orthe proteasome; any cell activation pathway including but not limited tothose involving protein kinase A, nitric oxide, caveolin-1, actin,calcium, protein kinase C, Cdc2, cyclin B, Cdc25, GRB2, SRC proteinkinase, ADP-ribosylation factors (ARFs), phospholipase D, AKAP95, p68,Aurora B, CDK1, Eg7, histone H3, PKAc, CD80, PI3 kinase, WASP, Arp2,Arp3, p16, p34, p20, PP2A, angiotensin, angiotensin-converting enzyme,protease-activated receptor-1, protease-activated receptor-4, Ras,RAF-1, PLCβ, PLCγ, COX-1, G-protein-coupled receptors, phospholipase A2,IP3, SUMO1, SUMO 2/3, ubiquitin, Ran, Ran-GAP, Ran-GEF, p53,glucocorticoids, glucocorticoid receptor, components of the SWI/SNFcomplex, RanBP1, RanBP2, importins, exportins, RCC1, CD40, CD40 ligand,p38, IKKα, IKKβ, NF_(K)B, TRAF2, TRAF3, TRAF5, TRAF6, IL-4, IL-4receptor, CDK5, AP-1 transcription factor, CD45, CD4, T cell receptors,MAP kinase, nerve growth factor, nerve growth factor receptor, c-Jun,c-Fos, Jun kinase, GRB2, SOS-1, ERK-1, ERK, JAK2, STAT4, IL-12, IL-12receptor, nitric oxide synthase, TYK2, IFNγ, elastase, IL-8, epithelins,IL-2, IL-2 receptor, CD28, SMAD3, SMAD4, TGFβ or TGFβ receptor; any cellcycle regulation, signaling or differentiation pathway including but notlimited to those involving TNFs, SRC protein kinase, Cdc2, cyclin B,Grb2, Sos-1, SHC, p68, Aurora kinases, protein kinase A, protein kinaseC, Eg7, p53, cyclins, cyclin-dependent kinases, neural growth factor,epidermal growth factor, retinoblastoma protein, ATF-2, ATM, ATR, AKT,CHK1, CHK2, 14-3-3, WEE1, CDC25 CDC6, Origin Recognition Complexproteins, p15, p16, p27, p21, ABL, c-ABL, SMADs, ubiquitin, SUMO, heatshock proteins, Wnt, GSK-3, angiotensin, p73 any PPAR, TGFα, TGFβ, p300,MDM2, GADD45, Notch, cdc34, BRCA-1, BRCA-2, SKP1, the proteasome, CUL1,E2F, p107, steroid hormones, steroid hormone receptors, I_(K)Bα,I_(K)Bβ, Sin3A, heat shock proteins, Ras, Rho, ERKs, IKKs, PI3 kinase,Bcl-2, Bax, PCNA, MAP kinases, dynein, RhoA, PKAc, cyclin AMP, FAK,PIP2, PIP3, integrins, thrombopoietin, Fas, Fas ligand, PLK3, MEKs,JAKs, STATs, acetylcholine, paxillin calcineurin, p38, importins,exportins, Ran, Rad50, Rad51, DNA polymerase, RNA polymerase, Ran-GAP,Ran-GEF, NuMA, Tpx2, RCC1, Sonic Hedgehog, Crm1, Patched (Ptc-1), MPF,CaM kinases, tubulin, actin, kinetochore-associated proteins,centromere-binding proteins, telomerase, TERT, PP2A, c-MYC, insulin, Tcell receptors, B cell receptors, CBP, IKB, NF_(K)B, RAC1, RAF1, EPO,diacylglycerol, c-Jun, c-Fos, Jun kinase, hypoxia-inducible factors,GATA4, β-catenin, α-catenin, calcium, arrestin, survivin, caspases,procaspases, CREB, CREM, cadherins, PECAMs, corticosteroids,colony-stimulating factors, calpains, adenylyl cyclase, growth factors,nitric oxide, transmembrane receptors, retinoids, G-proteins, ionchannels, transcriptional activators, transcriptional coactivators,transcriptional repressors, interleukins, vitamins, interferons,transcriptional corepressors, the nuclear pore, nitrogen, toxins,proteolysis, or phosphorylation; or any metabolic pathway including butnot limited to those involving the biosynthesis of amino acids,oxidation of fatty acids, biosynthesis of neurotransmitters and othercell signaling molecules, biosynthesis of polyamines, biosynthesis oflipids and sphingolipids, catabolism of amino acids and nutrients,nucleotide synthesis, eicosanoids, electron transport reactions,ER-associated degradation, glycolysis, fibrinolysis, formation of ketonebodies, formation of phagosomes, cholesterol metabolism, regulation offood intake, energy homeostasis, prothrombin activation, synthesis oflactose and other sugars, multi-drug resistance, biosynthesis ofphosphatidylcholine, the proteasome, amyloid precursor protein, RabGTPases, starch synthesis, glycosylation, synthesis of phoshoglycerides,vitamins, the citric acid cycle, IGF-1 receptor, the urea cycle,vesicular transport, or salvage pathways. It is further contemplatedthat nucleic acids molecules of the invention can be employed indiagnostic and therapeutic methods with respect to any of the abovepathways or factors. Thus, in some embodiments of the invention, asynthetic miRNA, nonsynthetic nucleic acid, or miRNA inhibitor inhibits,eliminate, activates, induces, increases, or otherwise modulates one ormore of the above pathways or factors is contemplated as part of methodsof the invention. The nucleic acid can be used to diagnosis a disease orcondition based on the relation of that miRNA to any of the pathwaysdescribed above.

Phenotypic traits also include characteristics such as longevity,appearance (e.g., baldness, obesity), strength, speed, endurance,fertility, and susceptibility or receptivity to particular drugs ortherapeutic treatments. Synthetic miRNAs or miRNA inhibitors that affectphenotypic traits may provide intervention points for therapeuticdevelopment.

H. Other Assays

In addition to the use of arrays and microarrays, it is contemplatedthat a number of difference assays could be employed to analyze miRNAs,their activities, and their effects. Such assays include, but are notlimited to, RT-PCR, in situ hybridization, hybridization protectionassay (HPA) (GenProbe), branched DNA (bDNA) assay (Chiron), rollingcircle amplification (RCA), single molecule hybridization detection (USGenomics), Invader assay (ThirdWave Technologies), and Bridge LitigationAssay (Genaco). It is contemplated that such methods may be used in thecontext of arrays, as well as in the context of diagnostic assays.

III. Therapeutic and Diagnostic Applications

Synthetic miRNAs or miRNA inhibitors that affect phenotypic traitsprovide intervention points for therapeutic applications as well asdiagnostic applications (by screening for the presence or absence of aparticular miRNA). It is specifically contemplated that RNA molecules ofthe present invention can be used to treat any of the diseases orconditions discussed in the previous section. Moreover, any of themethods described above can also be employed with respect to therapeuticand diagnostic aspects of the invention. For example, methods withrespect to detecting miRNAs or screening for them can also be employedin a diagnostic context.

In therapeutic applications, an effective amount of the synthetic miRNAsor miRNA inhibitors of the present invention is administered to a cell,which may or may not be in an animal. In some embodiments, atherapeutically effective amount of the synthetic miRNAs or miRNAinhibitors of the present invention is administered to an individual forthe treatment of disease or condition. The term “effective amount” asused herein is defined as the amount of the molecules of the presentinvention that are necessary to result in the desired physiologicalchange in the cell or tissue to which it is administered. The term“therapeutically effective amount” as used herein is defined as theamount of the molecules of the present invention that achieves a desiredeffect with respect to a disease or condition. A skilled artisan readilyrecognizes that in many cases the molecules may not provide a cure butmay provide a partial benefit, such as alleviation or improvement of atleast one symptom. In some embodiments, a physiological change havingsome benefit is also considered therapeutically beneficial. Thus, insome embodiments, an amount of molecules that provides a physiologicalchange is considered an “effective amount” or a “therapeuticallyeffective amount.”

In some embodiments the molecule has a sequence that corresponds to themiRNA sequence from that particular animal, as opposed to from anotheranimal. Thus, in some embodiments, a human sequence is utilized in theRNA molecules of the present invention.

A. Modes of Administration and Formulations

The nucleic acid molecules of the invention may be administered to asubject alone or in the form of a pharmaceutical composition for thetreatment of a condition or disease. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the proteins into preparations which can beused pharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

For topical administration the proteins of the invention may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art. Systemic formulations include those designedfor administration by injection, e.g. subcutaneous, intravenous,intramuscular, intrathecal or intraperitoneal injection, as well asthose designed for transdermal, transmucosal, inhalation, oral orpulmonary administration. For injection, the nucleic acids of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiological saline buffer. The solution may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the nucleic acid molecules may be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art. For oral administration,the nucleic acids can be readily formulated by combining the moleculeswith pharmaceutically acceptable carriers well known in the art. Suchcarriers enable the nucleic acids of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a patient to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, e.g.lactose, sucrose, mannitol and sorbitol; cellulose preparations such asmaize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulatingagents; and binding agents. If desired, disintegrating agents may beadded, such as the cross-linked polyvinylpyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. If desired, solid dosageforms may be sugar-coated or enteric-coated using standard techniques.For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added. For buccaladministration, the molecules may take the form of tablets, lozenges,etc. formulated in conventional manner. For administration byinhalation, the molecules for use according to the present invention areconveniently delivered in the form of an aerosol spray from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the nucleic acids and a suitable powder basesuch as lactose or starch. The RNA molecules may also be formulated inrectal or vaginal compositions such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides.

In addition to the formulations described previously, the molecules mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, themolecules may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well-known examples of delivery vehiclesthat may be used to deliver nucleic acids of the invention.

A nucleic acid of the invention may be administered in combination witha carrier or lipid to increase cellular uptake. For example, theoligonucleotide may be administered in combination with a cationiclipid. Examples of cationic lipids include, but are not limited to,lipofectin, DOTMA, DOPE, and DOTAP. The publication of WO0071096, whichis specifically incorporated by reference, describes differentformulations, such as a DOTAP:cholesterol or cholesterol derivativeformulation that can effectively be used for gene therapy. Otherdisclosures also discuss different lipid or liposomal formulationsincluding nanoparticles and methods of administration; these include,but are not limited to, U.S. Patent Publication 20030203865,20020150626, 20030032615, and 20040048787, which are specificallyincorporated by reference to the extent they disclose formulations andother related aspects of administration and delivery of nucleic acids.Methods used for forming particles are also disclosed in U.S. Pat. Nos.5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901, 6,200,801, and5,972,900, which are incorporated by reference for those aspects.

The nucleic acids may also be administered in combination with acationic amine such as poly (L-lysine). Nucleic acids may also beconjugated to a chemical moiety, such as transferrin and cholesteryls.In addition, oligonucleotides may be targeted to certain organelles bylinking specific chemical groups to the oligonucleotide. For example,linking the oligonucleotide to a suitable array of mannose residues willtarget the oligonucleotide to the liver.

Additionally, the molecules may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid polymers containing thetherapeutic agent. Various of sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the molecules for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the chimericmolecules, additional strategies for molecule stabilization may beemployed.

Nucleic acids may be included in any of the above-described formulationsas the free acids or bases or as pharmaceutically acceptable salts.Pharmaceutically acceptable salts are those salts that substantiallyretain the biologic activity of the free bases and which are prepared byreaction with inorganic acids. Pharmaceutical salts tend to be moresoluble in aqueous and other protic solvents than are the correspondingfree base forms.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more synthetic miRNA molecules or miRNAinhibitors dissolved or dispersed in a pharmaceutically acceptablecarrier. The phrases “pharmaceutical or pharmacologically acceptable”refers to molecular entities and compositions that do not produce anadverse, allergic or other untoward reaction when administered to ananimal, such as, for example, a human, as appropriate. The preparationof an pharmaceutical composition that contains at least one chimericpolypeptide or additional active ingredient will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The chimeric molecules may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present invention can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, inhalation (e.g. aerosolinhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The molecules may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments, the molecules are prepared for administration bysuch routes as oral ingestion. In these embodiments, the solidcomposition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

Any embodiment discussed above with respect to delivery or transport tocells can also be employed with respect to implementing delivery ofmedicinal compounds discussed in this section.

B. Effective Dosages

The molecules of the invention will generally be used in an amounteffective to achieve the intended purpose. For use to treat or prevent adisease condition, the molecules of the invention, or pharmaceuticalcompositions thereof, are administered or applied in a therapeuticallyeffective amount. A therapeutically effective amount is an amounteffective to ameliorate or prevent the symptoms, or prolong the survivalof, the patient being treated. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the molecules which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5to 1 mg/kg/day. Therapeutically effective serum levels may be achievedby administering multiple doses each day.

In cases of local administration or selective uptake, the effectivelocal concentration of the proteins may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

The amount of molecules administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The therapy may be repeated intermittently while symptoms detectable oreven when they are not detectable. The therapy may be provided alone orin combination with other drugs or treatment (including surgery).

C. Toxicity

Preferably, a therapeutically effective dose of the molecules describedherein will provide therapeutic benefit without causing substantialtoxicity.

Toxicity of the molecules described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD50 (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Proteinswhich exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (See,e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics,Ch. 1, p. 1).

D. Pendant Groups

A “pendant group” may be attached or conjugated to the nucleic acid.Pendant groups may increase cellular uptake of the nucleic acid. Pendantgroups can be linked to any portion of the nucleic acid but are commonlylinked to the end(s) of the oligonucleotide chain. Examples of pendantgroups include, but are not limited to: acridine derivatives (i.e.2-methoxy-6-chloro-9-aminoacridine); cross-linkers such as psoralenderivatives, azidophenacyl, proflavin, and azidoproflavin; artificialendonucleases; metal complexes such as EDTA-Fe(II),o-phenanthroline-Cu(I), and porphyrin-Fe(II); alkylating moieties;nucleases such as amino-1-hexanolstaphylococcal nuclease and alkalinephosphatase; terminal transferases; abzymes; cholesteryl moieties;lipophilic carriers; peptide conjugates; long chain alcohols; phosphateesters; amino; mercapto groups; radioactive markers; nonradioactivemarkers such as dyes; and polylysine or other polyamines. In oneexample, the nucleic acid is conjugated to a carbohydrate, sulfatedcarbohydrate, or glycan.

IV. Kits

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, individual synthetic miRNAs are included in a kit.The kit may further include one or more negative control syntheticmiRNAs that can be used to control for the effects of synthetic miRNAdelivery. The kit may further include water and hybridization buffer tofacilitate hybridization of the two strands of the synthetic miRNAs. Thekit may also include one or more transfection reagent(s) to facilitatedelivery of the synthetic miRNA to cells.

In another non-limiting example, multiple synthetic miRNAs and/ormultiple miRNA inhibitors are included in a kit. The kit may furtherinclude one or more negative control synthetic miRNAs and/or miRNAinhibitors that can be used to control for the effects of syntheticmiRNA and/or miRNA inhibitor delivery. The kit may also include one ormore transfection reagents to facilitate delivery into cells.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit (labelingreagent and label may be packaged together), the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a vial. The kits of thepresent invention also will typically include a means for containing thenucleic acids, and any other reagent containers in close confinement forcommercial sale. Such containers may include injection or blow-moldedplastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which thenucleic acid formulations are placed, preferably, suitably allocated.The kits may also comprise a second container means for containing asterile, pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Such kits may also include components that preserve or maintain themiRNA or that protect against its degradation. Such components may beRNAse-free or protect against RNAses. Such kits generally will comprise,in suitable means, distinct containers for each individual reagent orsolution.

A kit will also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionsmay include variations that can be implemented.

Kits of the invention may also include one or more of the following:synthetic miRNA, nonsynthetic miRNA, library of synthetic miRNAs,library of miRNA inhibitors, library of nonsynthetic miRNA, combinationlibrary of synthetic miRNA, miRNA inhibitors, and/or nonsyntheticmiRNAs, negative control synthetic miRNA, negative control miRNAinhibitor, negative control nonsynthetic miRNA, nuclease-free water;RNase-free containers, such as 1.5 ml tubes; hybridization buffer; andtransfection reagent(s).

It is contemplated that such reagents are embodiments of kits of theinvention. Such kits, however, are not limited to the particular itemsidentified above and may include any reagent used for the manipulationor characterization of miRNA.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Unless otherwise designated, catalog numbers refer to products availableby that number from Ambion, Inc.®, The RNA Company.

Example 1 Assay for Measuring Activity of Precursor miRNAs (Reporter)

A series of luciferase reporter vectors was created to measure theactivities of synthetic miRNAs in cells. The reporter vectors were basedon plasmids that had been used to monitor the activity of endogenousmiRNAs (Tuschl paper). Briefly, a mammalian expression vector with theluciferase gene under the control of the CMV early promoter was created.Down-stream of the luciferase coding sequence, in the 3′ UTR of thegene, sequences complementary to mature miR-1-2, miR-10, miR-124,miR-19a, and miR-130 were added. The reporter vectors wereco-transfected into HeLa cells along with synthetic miRNAs designed tointroduce one of the five miRNAs listed above. The transfectionsinvolved mixing 200 ng of reporter vector with 0.3, 1, and 3 pmoles ofeach corresponding synthetic miRNA. The reporter/miRNA mixture was mixedwith 0.3 μl of Lipofectamine 2000 (Invitrogen) and incubated for 5-15minutes. Approximately 8,000 cells were added to eachmiRNA/reporter/transfection reagent complex in individual wells of a96-well plate. HeLa cells were grown in D-MEM (GIBCO) supplemented with10% fetal bovine serum (GIBCO) at 37° C. and 5% CO₂. 24-48 hrs posttransfection, the cells were harvested and assayed using the Luciferaseassay as described by the manufacturer (Promega). The level ofluciferase expression in the cell populations was compared to cellstransfected with the same reporter but a synthetic miRNA with a sequencethat does not correspond to the vector. This non-targeting miRNA wasreferred to as the negative control miRNA.

Final analysis of the synthetic miRNA designs involved measuring theactivity of both the active and complementary strands of our syntheticmiRNAs. For these studies, reporter vectors with luciferase 3′ UTRsequences were created that included regions complementary to both theactive and the complementary strands of our synthetic miR-33 and let-7bmiRNA designs. When co-transfected with malfunctioning synthetic miRNAs,the reporters with a sequence targeted by the complementary strandexhibit reduced luciferase expression because the complementary strandof the synthetic miRNAs are entering the miRNA pathway in addition to oreven instead of the active strand that is desired. For theseexperiments, the co-transfection and reporter analysis protocols areidentical to what is described above.

Example 2 Assay for Measuring Activity of Precursor miRNAs (EndogenousGene)

While the luciferase reporter constructs were extremely valuable inevaluating the synthetic miRNA designs, it was important to verify thefindings of the reporter constructs by measuring the effects of thesynthetic miRNAs on endogenous gene targets. For these studies, theexpression of RAS and MYC in cells transfected with let-7 miRNAs waschosen for monitoring. Both RAS and MYC are down-regulated by thevarious members of the let-7 family in humans and C. elegans(publication pending). Using a microarray system specific to miRNAs, theinventors have found that HepG2 cells express undetectable levels oflet-7. To test the activities of our various designs of our syntheticmiRNAs, synthetic let-7 miRNAs were created and used to transfect HepG2cells in 24-well plates using siPORT NeoFX (Ambion) according to themanufacturer's suggestions. Three days post-transfection, the cells werefixed with 4% paraformaldehyde, stained with DAPI to localize cellnuclei, and stained with FITC-conjugated antibodies specific to MYC orRAS (US Biological) according to the manufacturer's suggestions. Therelative reduction in target protein expression in synthetic let-7transfected cells was determined by comparing the staining intensity ofMYC and RAS to cells transfected with a negative control miRNA usingMetaMorph software.

To ensure that the results of our let-7 assays could be verified byadditional miRNA interactions that are observed naturally in cells, wecreated assays for two additional miRNAs with verified targets. In thefirst, a real-time PCR™ assay was developed to measure the level of theHOXB8 mRNA in cells transfected with synthetic miR-196. It has beenshown that miR-196 induces degradation of the HOXB8 mRNA in cells. Whentransfected into cultured cells using siPORT NeoFX according to themanufacturer's instructions, effective miR-196 synthetic miRNA designsreduce the levels of the HOXB8 mRNA.

To monitor the effectiveness of synthetic miR-1-2 miRNAs, a reportervector was created wherein the 3′ UTR of the G6PD gene was placedimmediately down-stream of the luciferase coding region. An interactionbetween miR-1-2 and the G6PD 3′ UTR has been published (Lewis, 2003).Synthetic miR-1-2 designs were co-transfected with the reporter vectorand assayed as described in Example 1.

Example 3 Effectiveness of Partially Complementary miRNAs

Three general sequence designs were compared for miRNA activity. Thefirst, referred to as the “miRNA design,” featured an active strandidentical to the mature miRNA found in animals and a complementarystrand that was identical to the hairpin sequence that is predicted toexist in cells during the processing of the miRNA prior to activation ofthe miRNA (see below). The second design, referred to as the “mismatchdesign,” was a hybrid of the same active strand as above with acomplementary strand with a di-nucleotide, 3′ overhang and twomismatches in the final five nucleotides that preceded the 3′ overhang(see below). The third design, referred to as the “siRNA design,”comprised the same active strand as above hybridized to a second RNAthat was fully complementary except that it left 3′ di-nucleotideoverhangs at either end of the double-stranded molecule (twopolynucleotides) (see below). The examples below involve or correspondto human miRNAs.

miR-1-2 mature miR-1-2 sequence-UGGAAUGUAAAGAAGUAUGUA (53-73 of SEQ IDNO:1) miRNA design = CAUACUUCUUUAUAUGCCCAUA (SEQ ID NO:594) +UGGAAUGUAAAGAAGUAUGUA (SEQ ID NO:595) mismatch design =CAUACUUCUUUACAUUCUGTT (SEQ ID NO:596) + UGGAAUGUAAAGAAGUAUGUA (SEQ IDNO:597) siRNA design = CAUACUUCUUUACAUUCCATT (SEQ ID NO:598) +UGGAAUGUAAAGAAGUAUGUA (SEQ ID NO:599) mir-124a-1 mature miR-124sequence-UUAAGGCACGCGGUGAAUGCCA (52-73 of SEQ ID NO:80) miRNA design =GUGUUCACAGCGGACCUUGAUU (SEQ ID NO:600) + UUAAGGCACGCGGUGAAUGCCA (SEQ IDNO:601) mismatch design = GCAUUCACCGCGUGCCUUGGTT (SEQ ID NO:602) +UUAAGGCACGCGGUGAAUGCCA (SEQ ID NO:603) siRNA design =GCAUUCACCGCGUGCCUUAATT (SEQ ID NO:604) + UUAAGGCACGCGGUGAAUGCCA (SEQ IDNO:605) miR-130a mature miR-130 sequence-CAGUGCAAUGUUAAAAGGGC (55-74 ofSEQ ID NO:91) miRNA design = UCUUUUCACAUUGUGCUAC (SEQ ID NO:606) +CAGUGCAAUGUUAAAAGGGC (SEQ ID NO:607) mismatch design =UAUUUUAACAUUGCACUGTT (SEQ ID NO:608) + CAGUGCAAUGUUAAAAGGGC (SEQ IDNO:609) siRNA design = CCUUUUAACAUUGCACUGTT (SEQ ID NO:610) +CAGUGCAAUGUUAAAAGGGC (SEQ ID NO:611) miR-19a mature miR-19asequence-UGUGCAAAUCUAUGCAAAACUGA (49-71 of SEQ ID NO:28) miRNA design =AGUUUUGCAUAGUUGCACUA (SEQ ID NO:612) + UGUGCAAAUCUAUGCAAAACUGA (SEQ IDNO:613) mismatch design = ACAUUUGCAUAGAUUUGCACATT (SEQ ID NO:614) +UGUGCAAAUCUAUGCAAAACUGA (SEQ ID NO:615) siRNA design =AGUUUUGCAUAGAUUUGCACATT (SEQ ID NO:616) UGUGCAAAUCUAUGCAAAACUGA (SEQ IDNO:617) mmu-miR-10a-1 (mouse) mature miR-10sequence-UACCCUGUAGAUCCGAAUUUGUG (22-44 of SEQ ID NO:212) miRNA design =CAAAUUCGUAUCUAGGGGAAUA (SEQ ID NO:6 18) + UACCCUGUAGAUCCGAAUUUGUG (SEQID NO:619) mismatch design = AGAAUUCGGAUCUACAGGGUATT (SEQ ID NO:620) +UACCCUGUAGAUCCGAAUUUGUG (SEQ ID NO:621) siRNA design =CAAAUUCGGAUCUACAGGGUATT (SEQ ID NO:622) + UACCCUGUAGAUCCGAAUUUGUG (SEQID NO:623) miR-33 mature miR-33 sequence-GUGCAUUGUAGUUGCAUUG (6-24 ofSEQ ID NO:57) miRNA = AUGUUUCCACAGUGCAUCA (SEQ ID NO:624) +GUGCAUUGUAGUUGCAUUG (SEQ ID NO:625) mismatch design =GUCCAACUACAAUGCACTT (SEQ ID NO:626) + GUGCAUUGUAGUUGCAUUG (SEQ IDNO:627) siRNA design = AUGCAACUACAAUGCACTT (SEQ ID NO:628) +GUGCAUUGUAGUUGCAUUG (SEQ ID NO:629) let-7b mature let-7bsequence-UGAGGUAGUAGGUUGUGUGGUU (6-27 of SEQ ID NO:6) miRNA design =CUAUACAACCUACUGCCUUCC (SEQ ID NO:630) + UGAGGUAGUAGGUUGUGUGGUU (SEQ IDNO:631) mismatch design = CCACACAACCUACUAUCUUATT (SEQ ID NO:632) +UGAGGUAGUAGGUUGUGUGGUU (SEQ ID NO:633) siRNA design =CCACACAACCUACUACCUCATT (SEQ ID NO:634) + UGAGGUAGUAGGUUGUGUGGUU (SEQ IDNO:635) miR-196-2 mature miR-196 sequence-UAGGUAGUUUCAUGUUGUUGG (7-27 ofSEQ ID NO:143) siRNA design = AACAACAUGAAACUACCUATT (SEQ ID NO:636) +UAGGUAGUUUCAUGUUGUUGG (SEQ ID NO:637) miRNA design =CAAAUUCGUAUCUAGGGGAAUA (SEQ ID NO:638) + UAGGUAGUUUCAUGUUGUUGG (SEQ IDNO:639) mismatch design = AAUAACAUGAAACUACCUATT (SEQ ID NO:640) +UAGGUAGUUUCAUGUUGUUGG (SEQ ID NO:641)The assorted mir-1-2, mmu-miR-10a-1, miR-19a, mir-124a-1, and mir-130asynthetic miRNAs were tested for their capacity to reduce the expressionof the reporter gene in vectors with appropriate miRNA target sitesusing the assay described in Example 1. All three designs were similarlycapable of down-regulating the appropriate reporter vectors.

To assess whether there were differences between the various miRNAdesigns ini their ability to affect the expression of endogenous genes,the following cells were transfected: HepG2 cells with three designs ofthe let-7 synthetic miRNAs, A549 with three designs of the miR-196synthetic miRNAs, and HeLa with the G6PD reporter vector and threedesigns of the miR-1-2 synthetic miRNA. As with the reporter vectors,all three synthetic miRNA designs proved capable of reducing theexpression of the target genes, though it is notable that the siRNAdesign performed most poorly.

As a final comparison of the three synthetic miRNA designs, syntheticmiRNAs were co-transfected with reporter vectors that included targetsites for the complementary strands of the synthetic miRNAs according tothe procedure described in Example 1. In this assay, it was apparentthat the siRNA design significantly affected the reporter vectors,indicating that the wrong strand of the miRNA was entering the miRNApathway (FIG. 3). Because the complementary strand might impact theexpression of genes that are not natural targets of the miRNA that isbeing studied, the siRNA design is inappropriate for effective syntheticmiRNAs.

Example 4 Effectiveness of Chemically 5′ End-Modified Synthetic miRNAs

Although the siRNA design proved problematic in that it exhibited a highrate of complementary strand uptake by the miRNA pathway, it did havethe advantage that it was easy to hybridize and easy to deliver tocells. For these reasons, ways to overcome the problems withcomplementary strand uptake were explored. The siRNA design was used totest the effects of chemical modifications at the 5′ ends of thesynthetic miRNAs. For these studies, several different complementarystrands were synthesized with unique 5′ ends. One featured fourdeoxyribose nucleotides at the 5′ end; one was a combination of fourdeoxyribose nucleotides at the 5′ end and a 5′ NH₂; one had a 5′ NH₂;one had a 5′ NHCOCH₃ (see below).

miR-33 mature miR-33 sequence-GUGCAUUGUAGUUGCAUUG (6-24 of SEQ ID NO:57)siRNA design = AUGCAACUACAAUGCACTT (SEQ ID NO:642) + GUGCAUUGUAGUUGCAUUG(SEQ ID NO:643) 5′ amino design = (NH₂)AUGCAACUACAAUGCACTT (SEQ IDNO:644) + GUGCAUUGUAGUUGCAUUG (SEQ ID NO:645) 5′ acetyl design =(CH₃OCNH)AUGCAACUACAAUGCACTT (SEQ ID NO:646) + GUGCAUUGUAGUUGCAUUG (SEQID NO:647) 5′ DNA design = dAdUdGdCAACUACAAUGCACTT (SEQ ID NO:648) +GUGCAUUGUAGUUGCAUUG (SEQ ID NO:649) 5′ amino DNA design =(NH₂)dAdUdGdCAACUACAAUGCACTT (SEQ ID NO:650) + GUGCAUUGUAGUUGCAUUG (SEQID NO:651) let-7b mature let-7b sequence-UGAGGUAGUAGGUUGUGUGGUU (6-27 ofSEQ ID NO:6) siRNA design = CCACACAACCUACUACCUCATT (SEQ ID NO:652) +UGAGGUAGUAGGUUGUGUGGUU (SEQ ID NO:653) 5′ amino design =NH₂CCACACAACCUACUACCUCATT (SEQ ID NO:654) + UGAGGUAGUAGGUUGUGUGGUU (SEQID NO:655) 5′ DNA design = dCdCdAdCACAACCUACUACCUCATT (SEQ ID NO:656) +UGAGGUAGUAGGUUGUGUGGUU (SEQ ID NO:657) 5′ amino DNA design =NH₂dCdCdAdCACAACCUACUACCUCATT (SEQ ID NO:658) + UGAGGUAGUAGGUUGUGUGGUU(SEQ ID NO:659) miR-1-2 mature miR-1-2 sequence-UGGAAUGUAAAGAAGUAUGUA(53-73 of SEQ ID NO:1) siRNA design = CAUACUUCUUUACAUUCCATT (SEQ IDNO:660) + UGGAAUGUAAAGAAGUAUGUA (SEQ ID NO:661) 5′ amino design =NH₂CAUACUUCUUUACAUUCCATT (SEQ ID NO:662) + UGGAAUGUAAAGAAGUAUGUA (SEQ IDNO:663) miR-124a-1 mature miR-124 sequence-UUAAGGCACGCGGUGAAUGCCA (52-73of SEQ ID NO:80) siRNA design = GCAUUCACCGCGUGCCUUAATT (SEQ ID NO:664) +UUAAGGCACGCGGUGAAUGCCA (SEQ ID NO:665) 5′ amino design =NH₂GCAUUCACCGCGUGCCUUAATT (SEQ ID NO:666) + UUAAGGCACGCGGUGAAUGCCA (SEQID NO:667) miR-130a mature miR-130 sequence-CAGUGCAAUGUUAAAAGGGC (55-74of SEQ ID NO:91) siRNA design = CCUUUUAACAUUGCACUGTT (SEQ ID NO:668) +CAGUGCAAUGUUAAAAGGGC (SEQ ID NO:669) 5′ amino design =NH₂CCUUUUAACAUUGCACUGTT (SEQ ID NO:670) + CAGUGCAAUGUUAAAAGGGC (SEQ IDNO:671) miR-10a-1 mature miR-10 sequence-UACCCUGUAGAUCCGAAUUUGUG (22-44of SEQ ID NO:212) siRNA design = CAAAUUCGGAUCUACAGGGUATT (SEQ IDNO:672) + UACCCUGUAGAUCCGAAUUUGUG (SEQ ID NO:673) 5′ amino design =NH₂CAAAUUCGGAUCUACAGGGUATT (SEQ ID NO:674) + UACCCUGUAGAUCCGAAUUUGUG(SEQ ID NO:675)The miR-33 and let-7b synthetic miRNAs were co-transfected into HeLa andHepG2 cells, respectively, with reporter vectors bearing target sitesfor the active and complementary strands of miR-33 and let-7b asdescribed in Example 1. Luciferase expression from the active andcomplementary strand-specific reporter vectors was measured according tothe manufacturer's (Promega) protocol. As shown in FIG. 3, the syntheticmiRNA designs with the 5′ NH₂ and 5′ NHCOCH₃ provided higher activestrand activity and significantly reduced complementary strand activityrelative to the unmodified, synthetic miRNAs. This is ideal forsynthetic miRNAs since the effects seen following transfection will bespecific to the activity of the active strand of the synthetic miRNA.Furthermore, the high efficacy of the 5′ modified designs will allowlower concentrations to be used for transfections and reduce toxicitythat is often observed when transfecting cells with higher amounts ofnucleic acid.

To confirm that the 5′ amino modification is superior to the standardsiRNA design for a broad set of synthetic miRNAs, the effectiveness ofboth synthetic miRNA designs was measured in cells co-transfected withreporter vectors with miRNA target sites. As seen in FIG. 4, the 5′ NH₂is reproducibly superior to the unmodified siRNA design.

Example 5 Effectiveness of Chemically Internally Modified SyntheticmiRNAs

The siRNA design was also used to test the effects of chemicalmodifications at internal domains within the complementary strand. Forthese studies, 2′-Me modifications were placed at various locationsalong the length of the complementary strand. Below is provided anexample of a series of synthetic miRNAs with chemically modifiedcomplementary strands.

(SEQ ID NO:676) miRNA Strand - 5′-UAU ACA AGA GAU GAA AUC CUC-3′Complementary Strands - Position 1 - 5′-GGA UUU CAU CUC UUG UAU AUt-3′Position 2 - 5′-GGA UUU CAU CUC UUG UAU AUt-3′ Position 3 - 5′-GGA UUUCAU CUC UUG UAU AUt-3′ Position 4 - 5′-GGA UUU CAU CUC UUG UAU AUt-3′Position 5 - 5′-GGA UUU CAU CUC UUG UAU AUt-3′ Position 6 - 5′-GGA UUUCAU CUC UUG UAU AUt-3′ Note - Positions that are 2′-O-Me are denoted inbold.

Synthetic miRNAs with the designs described above were tested for miRNAand complementary strand activity. Interestingly, complementary strandmodifications at positions 1 and 5 significantly reduced complementarystrand activity without altering the activity of the miRNA strand (FIG.3).

Example 6 Synthetic miRNA Library Screen for miRNAs that Influence CellProliferation

A hallmark of cancer is uncontrolled cell proliferation; cellproliferation assays are commonly used by researchers to study theinfluence of genes in oncogenesis. A cell proliferation assay was usedin conjunction with the miRNA inhibitor library to identify miRNAs thatinfluence cell proliferation.

The inventors transfected HeLa cells in triplicate with fifteendifferent synthetic miRNAs using siPORT NeoFX (Ambion) according to themanufacturer's instructions (FIG. 6). Transfected HeLa cells wereanalyzed using Alamar Blue (BioSource International, Inc., CA) at 24 hrintervals. Alamar Blue is a compound, that when reduced by cellularmetabolism, changes from a non-fluorescent blue color to a fluorescentred form that is easily quantified. The amount of Alamar Blue reduced isdirectly proportional to the cell number, providing a rapid method forassessing cell proliferation. To perform the assay, the Alamar Bluereagent was added into the tissue culture media at a 10% finalconcentration. The mixture was incubated for 3-6 hr in growth conditionsafter which fluorescence was quantified using a Spectra MaX™ GeminiXS™(Molecular Devices, Sunnyvale, Calif.). Cells transfected with syntheticmiR-124 and miR-106 exhibited significantly lower proliferation thannegative control-transfected samples, as well as samples transfectedwith the other synthetic miRNAs.

Example 7 MiRNAs Inhibitor Library Screen for miRNAs that Influence CellProliferation

A hallmark of cancer is uncontrolled cell proliferation. Cellproliferation assays are commonly used by researchers to study theinfluence of genes in oncogenesis. A cell proliferation assay was usedin conjunction with our miRNA inhibitor library to identify miRNAs thatinfluence cell proliferation.

Cells were transfected with a library of over 90 miRNA inhibitors toidentify miRNAs that are involved in cell growth. HeLa cells (8000cells/well of 96 well plate) were transfected in triplicate with 5pmoles of miRNA inhibitors using siPORT™ NeoFX™ (Ambion). The media waschanged 24 hrs after transfection. 72 hours post-transfection, we fixedcells with 4% paraformaldehyde, permiabilized with 0.1% TritonX 100, andstained with propidium iodide to look at total cell number. The plateswere scanned using the TTP labtech Acumen Explorer. Cell number wasplotted relative to cells transfected with a negative control miRNAinhibitor (FIG. 7). The red horizontal bars bracket normal variation incell proliferation (20% variation). Insets: Specific miRNA inhibitorsthat either increased cell proliferation (left arrow) or did not affectcell proliferation (right arrow) were used in a second round ofscreening. HeLa cells were transfected with these miRNA inhibitors andcells were fixed and stained with anti b-actin antibody and DAPI tovisualize cell morphology changes in response to specific miRNAfunction. Cells transfected with the miRNA inhibitor that increased cellproliferation show marked alteration in cell morphology (left inset) vs.normal morphology (right inset).

A group of nine miRNA inhibitors were identified that caused significantdecreases (miR 31, 150, 187, 125a, 190, 191, 193, 204 and 218) in cellgrowth and two miRNA inhibitors that caused a significant increase (miR24 and miR 21) in cell growth following transfection into HeLa cells(Table 4). MiRNA-31 inhibition also caused a distinct cellularmorphology. A relative cut off of 20% above and below 100% was chosen asgenes that were considered significantly changed. These resultsdemonstrate the ability of individual human miRNAs to regulate importantcellular processes. Furthermore, the diversity of the observed effectsdemonstrates the potential complexity of cellular outcomes ofmiRNA-mediated regulation of gene expression.

TABLE 4 MiRNAs that affect cell proliferation Relative Impact on CellmiRNA Proliferation miR-31 Up regulation miR-150 Up regulation miR-187Up regulation miR-125a Up regulation miR-190 Up regulation miR-191 Upregulation miR-193 Up regulation miR-204 Up regulation miR218 Upregulation miR-21 Down regulation miR-24 Down regulation

Example 8 Synthetic miRNA Library Screen for miRNAs that InfluenceApoptosis

Many diseases including cancer are characterized by an inability toinstitute programmed cell death, or apoptosis. A caspase 3/7 activityassay was used in conjunction with a library of synthetic miRNAs toidentify miRNAs that are involved in regulating apoptosis.

A library of eighteen synthetic miRNAs was used to transfect A549 cells(8000 cells/well of 96 well plate) in triplicate using siPORT™ NeoFX™(Ambion). Media was changed after 24 hrs and cells were visuallyinspected under a microscope to qualitatively inspect cell death 72hours after transfection. The cells were measured for apoptosis bymeasuring caspase 3 activity as follows: 1) Cells were washed once withPBS and frozen at −80° C. 2) Cells were lysed by adding 40 μl of coldlysis buffer (50 mM HEPES pH 7.2, 40 mM NaCl, 0.5% NP40, 0.5 mM EDTA) tothe wells and incubated for 20 min at 4° C. 3) Add 160 μl ICE buffer (50mM HEPES pH 7.4, 0.1% CHAPS, 0.1 mM EDTA, 10% sucrose)+5 mM DTTcontaining 20 μM DEVDafc substrate. 4) Measure fluorescence increase inone hour at 400 ex, 505 em.

Cells transfected with miR-1-2 and miR-33 synthetic miRNAs exhibitedreduced caspase 3/7 activity and cells transfected with miR-20 exhibitedmuch higher levels of apoptosis. These three miRNAs likely regulategenes that are involved in controlling apoptosis.

Example 9 Screen for miRNAs that Influence Cell Viability

miRNA inhibitors were also used to identify miRNAs that influence cellviability. A library of over 90 miRNA inhibitors was used to transfectA549 cells (8000 cells/well of 96 well plate) in triplicate usingsiPORT™ NeoFX™ (Ambion). Media was changed after 24 hrs and cells werevisually inspected under a microscope to qualitatively inspect celldeath 72 hours after transfection. Cells were trypsinized and stainedwith ViaCount Flex Reagent, which distinguishes between viable andnon-viable cells based on permeability of the DNA binding dyes in thereagent. Cells were analyzed using the Guava PCA-96 (Personal CellAnalysis).

Twenty-one miRNA inhibitors induced a significantly different ratio oflive to dead cells than did the negative control miRNA inhibitor (FIG.8). Twelve reduced cell viability and nine increased cell viability(Table 5). Interestingly, there was littleoverlap in the miRNAs thataffected cell viability in A549 cells and those that affected cellproliferation in HeLa cells, suggesting that different cells responddifferently to have reduced miRNA activities or cell viability and cellproliferation are not affected by the same cellular pathways.

TABLE 5 MiRNAs that affect cell viability Relative Impact on Cell miRNAViability miR-7 Down miR-19a Down miR-23 Down miR-24 Down miR-27a DownmiR-31 Down miR-32 Down miR-134 Down miR-140 Down miR-150 Down miR-192Down miR-193 Down miR-107 Up miR-133 Up miR-137 Up miR-152 Up miR-155 UpmiR-181a Up miR-191 Up miR-203 Up miR-215 Up

Example 10 Screen for miRNAs that Influence Apoptosis

Apoptosis is a natural cellular process that helps control cancer byinducing death in cells with oncogenic potential. Many oncogenesfunction by altering induction of apoptosis. To identify miRNAs thatparticipate in apoptosis, an apoptosis assay was used with the miRNAinhibitor library.

Using a library of over 90 miRNA inhibitors, we screened for miRNAs thataffect apoptosis. HeLa cells (8000 cells/well of 96 well plate) weretransfected in triplicate with miRNA inhibitors (5 pmoles) using AmbionsiPORT™ NeoFX™. The media was changed 24 hrs after transfection andprocessed cells 72 hours after transfection. The cells were measured forapoptosis by measuring caspase 3 activity as follows: 1) Cells werewashed once with PBS and frozen at −80° C. 2) Cells were lysed by adding40 μl of cold lysis buffer (50 mM HEPES pH 7.2, 40 mM NaCl, 0.5% NP40,0.5 mM EDTA) to the wells and incubated for 20 min at 4° C. 3) Add 160μl ICE buffer (50 mM HEPES pH 7.4, 0.1% CHAPS, 0.1 mM EDTA, 10%sucrose)+5 mM DTT containing 20 μM DEVDafc substrate. 4) Measurefluorescence increase in one hour at 400 ex, 505 em.

Samples were also analyzed for cell number using a general esteraseassay to normalize the caspase 3 results. FDA substrate (0.4 mg/mlfluorescein diacetate (FDA) in acetonitrile) was diluted 1:19 intodilution buffer (40 mM Tris Cl pH 7.5, 20 mM NaCl, 0.5% NP-40, 0.02mg/ml final conc). 40 μl buffer (40 mM TrisCl pH 7.5, 0.5% NP-40) wasadded to each sample well. Samples were incubated 10 min on ice. 160 μlof diluted FDA substrate was added to each well. Fluorescence wasmeasured for 30 min at 37° C. (ex=488, em=529). The slope offluorescence increase over time is a function of the cell number in theplate.

Normalized screening data are displayed in FIG. 9. miRNAs that affectapoptosis are listed in Table 6.

TABLE 6 MiRNAs that affect apoptosis Relative Impact on Cell miRNAProliferation miR-31 Down miR-214 Down miR-7 Up miR-1-2 Up miR-148 UpmiR-195 Up miR-196 Up miR-199a Up miR-204 Up miR-210 Up miR-211 UpmiR-212 Up miR-215 Up miR-216 Up miR-218 Up miR-296 Up miR-321 Up

Example 11 Expression Analyses Using Synthetic RNAs

In addition to using phenotypic assays to identify miRNAs that influencegross cellular processes or cellular pathways, collections of syntheticmiRNAs and/or miRNA inhibitors can be used to identify miRNAs thatdirectly regulate the expression of a gene. A plasmid was created thathad a luciferase gene immediately upstream of the 3′UTR of the G6PDgene. A549 cells were co-transfected with the reporter vector andeighteen different synthetic miRNAs. 24 hours post-transfection,luciferase activity in the various cell populations was measured.Interestingly, the miR-1-2 significantly reduced the expression of theluciferase/G6PD gene, indicating that this family of miRNAs regulatesthe expression of the G6PD gene. Similar experiments can be used toidentify miRNAs that regulate the expression of such important genes asp53, BRCA1 and BRCA2, RAS, MYC, BCL-2, and others.

Example 12 Oncogeneic miRNAs Differential Expression and CancerRegulation

As noted in previous examples, a number of miRNAs have been identifiedthat are differentially expressed between tumor and normal adjacenttissue samples from the same cancer patients. Interestingly, there issignificant overlap in the miRNAs that are differentially expressedbetween different cancers, suggesting there is a core set of miRNAs thatinfluence cellular processes that when altered, lead to cancer. Thefollowing describes experiments aimed at developing a link between miRNAmis-regulation and cancer.

miRNA Expression in Lung Cancer

Twenty-two tumor and normal adjacent tissue (NAT) samples from lungcancer patients were analyzed using the miRNA array system describedabove. The arrays were analyzed and the relative expression of eachmiRNA was compared between the tumor and normal adjacent tissues fromeach patient. The various miRNAs were clustered based on their relativeexpression in tumors across different patients (FIG. 14). Six miRNAs(miR-126, 30a, 143, 145, 188, and 331) were expressed at significantlylower levels in the tumors of more than 70% of the patients. Two miRNAs(miR-21 and 200b) were expressed at significantly higher levels in thetumors of more than 70% of the patients. The differential expression ofa number of these miRNAs was verified by Northern analysis (FIG. 15).

miRNA Expression in Colon Cancer

Twenty-five tumor and NAT samples from colon cancer patients wereanalyzed using our miRNA array process. Like the lung cancercomparisons, the various miRNAs were clustered based on their relativeexpression in tumors across the different colon cancer patients (FIG.14). Five miRNAs (miR-143, 145, 195, 130a, and miR-331) were expressedat significantly lower levels in the tumors of more than 70% of thepatients. Five miRNAs (miR-223, 21, 31, 17, and 106) were expressed atsignificantly higher levels in the tumors of more than 70% of thepatients.

miRNAs as Cancer Markers

It is interesting that eight different miRNAs were differentiallyexpressed between the tumor and normal adjacent samples for most of thelung and colon patient samples that we analyzed (FIG. 16). These samemiRNAs were also found to be differentially expressed in the breast,thymus, bladder, pancreatic, and prostate cancer patients that weanalyzed, suggesting that these miRNAs might control cellular processesthat when altered lead to cancer.

miRNAs as Regulators of Oncogene Expression

To address whether specific miRNAs might be participating in cancerthrough the mis-regulation of oncogenes, we scanned the 3′ untranslatedregions (UTRs) of 150 well-known oncogenes for sequences withsignificant homology to the miRNAs identified in our microarrayanalysis. Potential target sites were selected based on two criteria:

-   -   (1) Perfect complementarity between positions 2-9 of the miRNA        and the oncogene. This miRNA core sequence has been identified        as critical to the activities of miRNAs and the known miRNA        target sites have essentially 100% complementarity at this site        (Doench et al. 2004).    -   (2) Overall T_(m) of the miRNA/mRNA interaction. In addition to        the core sequence, overall binding stability between miRNAs and        mRNAs has been shown to be an important indicator of miRNA        activity (Doench et al., 2004).

As seen in Table 8, potential target sites in the 3′UTRs of knownoncogenes were identified for all of the miRNAs that were observed to beroutinely differentially expressed in tumor samples. Interestingly,KRAS2, MYCL1, and CBL have multiple predicted miRNA binding sites whichcould provide the cooperative miRNA binding that has been implicated asan important factor in miRNA regulation (Doench et al. 2003); Zeng etal., 2003). Many of the genes listed in Table 8 become oncogenic whenthey are over-expressed, thus it is conceivable that reduced expressionof a miRNA could lead to up-regulation of one or more oncogenes andsubsequently lead to oncogenesis.

TABLE 8 Cancer-related miRNAs and their putative oncogene targets miRNAPredicted Gene Target let-7 RAS let-7 C-MYC miR-21 mutS homolog 2 (MSH2)miR-21 v-ski sarcoma viral oncogene homolog (avian) (SKI) miR-143breakpoint cluster region (BCR) miR-143 MCF.2 cell line derivedtransforming sequence (MCF2) miR-143 von Hippel-Lindau tumor suppressor(VHL) miR-143 v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog(KRAS2) miR-143 v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog(KRAS2) miR-143 Cas-Br-M (murine) ecotropic retroviral transformingsequence (CBL) miR-143 Cas-Br-M (murine) ecotropic retroviraltransforming sequence (CBL) miR-145 v-myc myelocytomatosis viral relatedoncogene (MYCN) miR-145 fibroblast growth factor receptor 2 (FGFR2)miR-145 Cas-Br-M (murine) ecotropic retroviral transforming sequence(CBL) miR-188 v-myc myelocytomatosis viral oncogene homolog 1 (MYCL1)miR-200b cadherin 13 (CDH13) miR-200b v-kit Hardy-Zuckerman 4 felinesarcoma viral oncogene homolog (KIT) miR-219 v-myc myelocytomatosisviral oncogene homolog 1 (MYCL1) miR-219 B-cell CLL/lymphoma 2 (BCL2)miR-219 cadherin 1, type 1, E-cadherin (epithelial) (CDH1) miR-331 vav 1oncogene (VAV1) miR-331 fibroblast growth factor receptor 1 (FGFR1)miR-331 BCL2-antagonist/killer 1 (BAK1) miR-331 retinoic acid receptor,alpha (RARA) miR-331 v-src sarcoma (Schmidt-Ruppin A-2) viral oncogenehomolog (SRC)

Example 13 Measuring the Effect of miRNAs on Oncogene Expression

Confirming miRNA target site predictions can be done in a variety ofways. In Drosophila and C. elegans, genetic approaches have been appliedwherein mutations in the miRNA and the putative miRNA target site(s) aremade and shown to result in similar phenotypes (Ha et al., 1996; Vellaet al., 2004). In mammalian cells, where genetic approaches are far moredifficult, reporter constructs have been used to show that the 3′ UTRsof putative target genes are regulated in cells at levels that aredisproportionate to reporter vector controls that contain mutations inthe putative miRNA binding sites (Lewis et al. 2003). In addition,vectors and oligonucleotides have been used to introduce or inhibitmiRNAs in cells to determine the effects on endogenous levels ofputative target genes (Lewis et al., 2003; Kiriakidou et al. 2004). Thelatter approach has been undertaken to validate the miRNA target sitepredictions.

Synthetic miRNAs and miRNA inhibitors have been developed that can betransfected into mammalian cells to either introduce miRNAs into cellsor inhibit the activity of miRNAs in cells, respectively. See U.S. Ser.No. 60/627,171, which is hereby incorporated by reference. A syntheticmiRNA and a miRNA inhibitor corresponding to let-7b were used todetermine if the target site predictions were correct. In theseexperiments, cultured cells that express undetectable levels of themiRNA were transfected with the synthetic miRNA using siPORT™ NeoFX™Transfection Agent (Ambion). Immunofluorescence assays were used to RASand C-MYC in the transfected cells. The proteins from both oncogeneswere expressed at almost three-fold lower levels in cells transfectedwith the synthetic miRNA than cells transfected with a Negative ControlmiRNA (Ambion). In a reciprocal experiment, cells that naturally expresshigh levels of the miRNA were transfected with the let-7 miRNAinhibitor. As expected, the proteins from both oncogenes were higher incells transfected with the miRNA inhibitor than in cells transfectedwith the Negative Control inhibitor (Ambion). These results areconsistent with the model that the miRNA regulates the expression of thetwo oncogenes. These data suggest that mis-regulation of a key miRNAcould participate in cancer progression by failing to regulate theexpression of one or more oncogenes.

Example 14 miRNAs in Lupus

Systemic lupus erythematosus (SLE; Lupus) is a chronic inflammatoryauto-immune disease that ultimately leads to immune complex-mediatedend-organ failure. It is characterized by an over activation of CD4+ Thelper cells and repression of CD8+ T cytotoxic activity, leading to anoverproduction of natural antibodies and pathogenic autoantibodies.Recently several histone modifications were reported in peripheral bloodmononuclear cells (PBMCs) isolated from lupus patients. Diagnosis oflupus is still frequently incorrect mainly because the symptoms vary sowidely and they come and go frequently, and because the disease mimicsso many other disorders. Furthermore, diagnosis does not indicate theparticular therapy to be used. In the absence of a cure, present-daytreatment of lupus is still primarily tailored to symptomatic relief andnot to the diagnosis. A diagnostic assay with high specificity andsensitivity would be very important.

Samples were analyzed from 16 individuals, 8 with clinically verifiedlupus and 8 non-lupus patients that were age- and gender-matched withthe lupus patients. Total RNA from these samples was isolated using theglass fiber filter method described above. The total RNA wasfractionated by tube electrophoresis to recover the miRNAs. The miRNAswere fluorescently labeled with Cy3 or Cy5 using the two-stepfluorescent labeling process described above. The labeled miRNAs werehybridized to miRNA probes arrayed on glass slides as described above.The signal from the hybridized miRNAs was quantified using a GenePix4000B Scanner (Axon) and the lupus and normal sample signals werecompared to identify differentially expressed miRNAs. Each arrayexperiment included duplicate arrays.

Fourteen miRNAs were differentially expressed in all of the lupussamples relative to the matched samples. miR-301, miR-199, miR-95,miR-105, mu-miR-290, miR-215, miR-188, miR-186, miR-211, miR-331, andmiR-137 were expressed at 50% or less in the lupus samples than thecorresponding normal samples. miR-21, miR-223, and miR-342 wereexpressed at 50% or greater in the lupus samples than the correspondingnormal samples. Several of the miRNAs were differentially expressed byas much as ten-fold between the lupus and normal samples. These miRNAsrepresent targets for diagnostic assay of therapeutic development.

Example 15 miRNAs and Prion Diseases

Novel infectious particles, termed prions, composed largely and perhapssolely of a single protein, are the likely causative agents of a groupof transmissible spongiform encephalopathies that produce lethal declineof cognitive and motor function. Evidence indicates that the responsibleprotein arrives at a pathogenic state by misfolding from a normal formthat has ubiquitous tissue distribution.

Using two cell-based prion model systems, the identification of miRNAsthat might be associated with the process was pursued. One model systemcomprises two cell lines, one of which is susceptible to prion formationand one that is not. The second model system involves cells before andafter they have been infected with prions. Total RNA fromprion-sensitive cells, prion-insensitive cells, and prion-infected cellswas isolated using the glass fiber filter method described above. Thetotal RNA was fractionated by tube electrophoresis to recover themiRNAs. The miRNAs were fluorescently labeled with Cy3 or Cy5 using thetwo-step fluorescent labeling process described above. The labeledmiRNAs were hybridized to miRNA probes arrayed on glass slides asdescribed above. The signal from the hybridized miRNAs was quantifiedusing a GenePix 4000B Scanner (Axon) and the signal from each of thesamples was compared to identify differentially expressed miRNAs.

As seen in FIG. 13, ten miRNAs were significantly up- or down-regulatedin both prion-sensitive and prion-infected cells relative to prionresistant, uninfected cells. Arrays on multiple biological replicatesfor both model systems have confirmed these results. Based on theirexpression profiles, miR-95, 135a, 7, 9, 27a, 130a, 16, 26a, and 24likely are involved directly or indirectly in prion infection and mightrepresent diagnostic or therapeutic targets for prion disease.

Example 16 Stroke-Associated miRNAs

Stroke is a major cause of death and permanent disability in humans.They occur when blood flow to a region of the brain is obstructed andmay result in death of brain tissue. There are two main types of stroke:ischemic and hemorrhagic. Ischemic stroke is caused by blockage in anartery that supplies blood to the brain, resulting in a deficiency inblood flow (ischemia). Hemorrhagic stroke is caused by the bleeding ofruptured blood vessels (hemorrhage) in the brain. Understanding miRNAsinvolved in stroke might enhance detection and/or treatment.

A stroke model system was used wherein mice are “preconditioned” byreducing oxygen flow to the brain (Kitagawa 1991). An equivalent set ofsix mice were used; three were preconditioned and three were untreated.24 hours after pre-conditioning, the mice were sacrificed. Total RNAfrom these samples was isolated using the glass fiber filter methoddescribed above. The total RNA was fractionated by tube electrophoresisto recover the miRNAs. The miRNAs were fluorescently labeled with Cy3 orCy5 using the two-step fluorescent labeling process described above. Thelabeled miRNAs were hybridized to miRNA probes arrayed on glass slidesas described above. The signal from the hybridized miRNAs was quantifiedusing a GenePix 4000B Scanner (Axon) and the preconditioned and normalsample signals were compared to identify differentially expressedmiRNAs.

Analysis of the miRNA profiles of the preconditioned animals (labeledP1, P2, and P4) revealed 10 miRNAs that were expressed at significantlydifferent levels in all three pre-conditioned animals relative to thethree non-treated animals (FIG. 14). These miRNAs resulted from ischemicpre-conditioning and represent potential targets for stroke diagnosis,prevention, or treatment.

Example 16

Synthetic miRNA Library Screens for miRNAs that Influence CellProliferation and Cell Viability in Various Cell Types

A hallmark of cancer is uncontrolled cell proliferation; cellproliferation assays are commonly used by researchers to study theinfluence of genes in oncogenesis. A cell proliferation assay was usedin conjunction with the miRNA inhibitor library to identify miRNAs thatinfluence cell proliferation.

HeLa (human ovarian cancer) and A549 (human lung cancer) cells weretransfected in triplicate with 150 synthetic miRNAs using siPORT NeoFX(Ambion) according to the manufacturer's instructions. The 150 are asfollows: Let-7a, Let-7b, Let-7c, Let-7d, Let-7g, mir-1, mir-7, mir-9,mir-10a, mir-10b, mir-15a, mir-16, mir-18, mir-19a, mir-17-3p, mir-20,mir-21, mir-22, mir-23a, mir-23b, mir-24, mir-25, mir-26a, mir-27a,mir-28, mir-29a, mir-31, mir-32, mir-30a-3p, mir-34a, mir-92, mir-95,mir-96, mir-98, mir-99a, mir-100, mir-101, mir-103, mir-105, mir-107,mir-108, mir-122, mir-124, mir-125a, mir-125b, mir-126, mir-128,mir-129, mir-132, mir-133A, mir-133B, mir-134, mir-135, mir-136,mir-137, mir-139, mir-140, mir-141, mir-142, mir-143, mir-144, mir-145,mir-146, mir-147, mir-148, mir-149, mir-150, mir-151, mir-152, mir-153,mir-155, mir-181a, mir-182, mir-183, mir-184, mir-186, mir-187, mir-188,mir-190, mir-191, mir-192, mir-193, mir-194, mir-195, mir-196, mir-197,mir-198, mir-199, mir-201, mir-203, mir-204, mir-205, mir-206, mir-207,mir-208, mir-210, mir-211, mir-212, mir-214, mir-215, mir-216, mir-217,mir-218, mir-219, mir-220, mir-221, mir-223, mir-224, mir-299, mir-301,mir-302, mir-320, mir-322, mir-323, mir-325, mir-324-3p, mir-328,mir-330, mir-331, mir-335, mir-337, mir-338, mir-339, mir-340, mir-345,mir-346, mir-367, mir-368, mir-369, mir-370, mir-371, mir-372, mir-373,mir-374, mu-mir-290, mu-mir-291, mu-mir-292-3p, mu-mir-293, mu-mir-294,mu-mir-295, mu-mir-297, mu-mir-298, mu-mir-329, mu-mir-341, mu-mir-344,mu-mir-351, mu-mir-376b, mu-mir-380-3p, mu-mir-409, mu-mir-411,mu-mir-412

The synthetic miRNAs were double stranded nucleic acid moleculescomposed of an active strand and a complementary strand. The activestrand contained a sequence that was identical to the correspondingmature miRNA. The complementary strand contained a sequence that was100% complementary to the relevant region of the mature miRNA sequence,but 1) lacking two nucleotides on its 3′ end that were complementary tothe mature miRNA sequence (at the 5′ end of the active strand) and 2)having a dinucleotide overhang on its 5′ end with respect to the activestrand. In other words, the two strands were fully complementary to theother's sequence except that each strand has a dinucleotide 5′ overhangwith respect to the other strand. The same kind of synthetic miRNAs wereused for Examples 17-20 as well. Any exceptions are described below. ThemiRNAs indicated in the tables identify the miRNA that corresponds tothe provided synthetic sequence.

Jurkat cells (human leukemia cell) and primary human T-cells intriplicate were electroporated with the same set of synthetic miRNAsusing siPorter-96 (Ambion) according to the manufacturer's instruction.All cells were analyzed for viable and non-viable cells 72 hourspost-transfection using the PCA-96 (Guava) with the Viacount Assay.Viable cell number is the number of live cells in a well at the point ofthe assay. The numbers provided in the tables below are equal to theaverage number of viable cells in wells transfected with a particularmiRNA divided by the number of viable cells in wells transfected withnegative control synthetic miRNAs multiplied by 100 to yield the % CellViability of miRNA-transfected cells relative to negative controltransfected cells.

Significance was assigned based on the average values of the negativecontrol transfected samples. miRNAs that were significantly differentthan the negative controls were qualified as “significant” based onbeing at least two standard deviations above or below the negativecontrol data.

The sequence if miRNA-325 is 5′-ccuaguagguguccaguaagugu-3′,

TABLE 9 miRNAs That Significantly Reduce Cell Viability of HeLa Cells %Viability std dev mir-345 75 5.9 mir-346 77.8 8.2 mir-193 79.6 14.7mir-206 79.6 6.5 mir-337 80.8 3.1 mmu-mir-293 82.6 1.7 mir-299 84.0 4.0mmu-mir-329 84.5 4.5 mmu-mir-409 86 2.8 mmu-mir-292-3p 86.2 2.8 mir-21086.4 5.1 mmu-mir-344 86.4 5.3 mmu-mir-298 86.7 4.2 mir-208 87.4 4.5mir-197 87.6 7.5 mir-217 87.9 3.5 mir-1 88.2 9.0 mir-124 88.8 4.2

TABLE 10 miRNAs That Significantly Reduce Viable Cell Number of HeLaCells Total Cell std dev Let-7b 16.2 8.1 Let-7g 22.7 8.2 Let-7c 24.1 7.2mir-124 24.5 3.4 Let-7a 25.4 1.2 Let-7d 37.3 2.3 mir-337 37.5 16.9 mir-138.7 2.2 miR-299 38.9 4.2 mir-34a 40.5 13.3 mmu-mir-292 41.2 8.3 mir-12241.2 6.5 mir-346 41.9 4.3 mir-101 43.4 6.4 mir-210 47.1 8.4 mir-147 47.78.2 mir-98 50.6 2.6 mir-345 51.8 6.8 miR-92 52.4 6.8 miR-96 53.2 0.9mir-7 54.0 5.3 mir-133b 55.9 3.1 mir-206 56.0 12.4 mmu-mir-297 56.0 5.7mir-19a 57.2 20.6 mmu-mir-344 57.5 14.1 mir-205 58.9 18.7 mir-208 60.511.1

TABLE 11 miRNAs That Significantly Increase Viable Cell Number of HeLaCells Total Cell Std dev mir-32 142.9 25.4 mu-miR-290 143.5 17.6 mir-212143.5 10.4 mir-92 144.7 16.8 mir-323 147.3 25.9 mir-145 148.1 22.2mir-324 148.2 9.0 mir-198 152.1 67.8 mir-27a 156.2 13.4 mir-369 158.427.3 mir-31 159.3 16.1 mir-335 161.7 20.8 mmu-mir-351 162.3 6.9 mir-370164.3 4.5 mir-325 169.6 19.8 mir-331 172.5 24.0 mir-139 181.3 11.2

TABLE 12 miRNAs That Significantly Reduce Cell Viability of A549 Cells %Viability St dev mir-193 92.4 2.5 mir-224 92.5 1.4 mir-96 92.6 0.1mir-346 93.9 1.6 mmu-mir-293 94.9 0.7 mir-34a 95 0.2 mir-216 95.1 1.0mmu-mir-380 95.2 0.8 mir-182 95.6 0.8 mir-301 95.6 1.0 mmu-mir-344 95.80.2 mmu-mir-409 95.8 0.6 mir-369 95.9 0.7

TABLE 13 miRNAs That Significantly Reduce Viable Cell Number in A549Cells Cell Number St Dev mir-124 44.3 2.2 mir-16 52.9 1.3 mir-337 54.77.0 mir-195 59.3 6.7 mir-34a 60.8 2.1 mir-15a 60.9 3.7 mir-28 61.3 0.8Let-7g 61.9 0.8 mmu-mir-292 62.2 2.3 mmu-mir-344 62.6 9.1 mir-7 62.9 4.6mir-193 63.7 3.3 mir-137 63.9 1.3 mir-147 64.8 0.5 mir-29a 67.0 3.8mir-129 67.2 3.3 mir-22 67.5 3.4 mir-126 68.0 2.6 mir-345 69.2 7.4mir-192 69.5 5.9 Let-7b 70.2 2.2 Let-7d 70.5 2.7 mir-346 70.9 7.1

TABLE 14 miRNAs That Significantly Increase Viable Cell Number in A549Total cell Std dev mir-373 110.4 7.9 mir-25 111.8 6.0 mmu-mir-294 112.15.9 mir-32 120.8 4.3 mir-92 122.4 4.0

TABLE 15 miRNAs That Significantly Reduce Cell Viability of JurkatsCells % Viability St Dev let-7a 20.54 0.70 miR-10b 35.98 2.92 let-7b48.79 5.08 miR-17-3p 61.55 15.63 miR-30a-3p 64.36 26.60 miR-34a 65.4520.44 miR-122 65.63 17.80 miR-29a 66.44 7.14 miR-101 67.44 29.56miR-133a 71.51 17.82 miR-19a 71.77 23.79 miR-32 75.59 11.69 miR-1 75.7412.92 miR-132 76.32 16.22 miR-28 77.07 16.58 miR-20 77.60 15.23 miR-13478.96 1.75

TABLE 16 miRNAs That Significantly Increase Cell Viability in JurkatCells Total cell Std dev miR-181-a 122.77 22.40 miR-9 124.63 9.98miR-141 126.08 24.03 miR-98 126.24 11.90 miR-10a 126.86 8.93 miR-125b128.71 3.50 miR-126 130.69 18.20 miR-100 130.77 14.60 miR-23b 132.183.50 miR-140 135.73 4.08 miR-155 142.57 22.40 miR-15a 143.01 11.29miR-129 146.94 9.92 miR-25 150.25 17.85 miR-143 158.74 1.86 miR-26a166.09 13.65

TABLE 17 miRNAs that Significantly Reduce Cell Viability in PrimaryT-Cells % Viability St Dev miR-184 61.04 12.16 miR-145 68.98 11.23miR-186 69.64 6.99 miR-139 69.85 0.29 miR-134 71.90 22.42 miR-190 75.592.43 miR-144 77.13 4.18 miR-183 77.71 2.86 miR-147 78.09 0.33 miR-14078.70 5.81 miR-155 79.26 10.68

TABLE 18 miRNAs that Significantly Increase Cell Viability of PrimaryT-Cells % Viability St Dev miR-126 120.81 40.08 miR-10b 121.28 18.86miR-17 122.46 3.71 miR-10a 124.11 9.46 miR-20 124.75 13.60 let-7c 124.814.00 miR-125a 125.66 5.13 miR-15a 129.07 10.96 let-7b 130.11 13.48let-7a 130.88 16.16 miR-18 131.73 1.75

It is interesting to note that the miRNAs that affect one cell typeoften fail to affect other cell types. This is likely due to the factthat the cellular processes that are active vary between different celltypes. This can be vitally important when considering the potential ofmiRNA-based therapeutics. Abnormal (disease) cells are different fromnormal cells owing to the fact that different cellular processes areactive in the two cell types. Identifying miRNAs that have differentialeffects on normal and abnormal cells would be ideal since they could bedelivered globally and expected to have an effect on only disease cells.When the cell viability data were compared for the leukemia (cancerousT-cell) cells and primary T-cells, it was noted that let-7a, let-7b, andmiR-10b all significantly reduce the percentage of viable cells in theleukemia cells while essentially having no effect on the correspondingnormal T-cells. These miRNAs are candidates for leukemia drugs.

Example 17 Synthetic miRNA Library Screen for miRNAs that Influence ERKActivation

In order for cancer cells to proliferate they must subvert both themachinery that controls the cell division cycle and the process ofprogrammed cell death (apoptosis). This is frequently achieved bymutation of specific proto-oncogenes such as Ras or tumor suppressorssuch as p53. The Ras-family of membrane associated GTPases transmitsignals into the interior of the cell by the activation of a number ofcytosolic signal transduction pathways such as the Raf>MEK>ERK MAPkinase signaling pathway. Disregulation of the Ras/Raf/MEK/ERK pathwayplays a major role in cancer pathogenesis (Meijer).

To identify miRNAs that affect ERK activation, HeLa cells weretransfected in a 96-well plate format with 150 different syntheticmiRNAs. Prior to transfection, the HeLa cells were trypsinized to removeadherent cells and diluted in normal growth medium to 10⁵ cells/mL. 0.5μl of siPort NeoFX in 9.5 μl of Optimem I medium was added to the cellsand incubated for 10 minutes at room temp (10 μL for each sample).miRNAs were rehydrated with 10 μl of diluted siPORT NeoFX. The sampleswere incubated at 37° C. and then the transfected samples were evaluated72 hours after transfection.

The controls for ERK activation were performed by depriving the wells ofa phosphate source for detection of ERK phosphorylation. 100 μl ofserum-free media (DMEM) to 37° C. was added per well and the cells wereincubated for 4 hours at 37° C. to attain basal phosphorylation levels.For the positive control wells, serum-free media was aspirated fromwells and 100 μL of 100 ng/mL EGF was added before incubating the cellsfor 7.5 minutes at 37° C.

Media from all wells was removed by aspiration and the cells wereimmediately fixed in 150 μL of 3.7% Formaldehyde in 1×PBS for 20 minutesat room temp with no shaking. Fixing solution was removed to anappropriate waste container. The fixed cells were washed three timeswith 1×PBS. The wells were then washed three times with 200 μL of 1×PBScontaining 0.1% Triton X-100 for 5 minutes per wash, with shaking atroom temp.

Cells were blocked by adding 150 μL of Li—COR Odyssey Blocking Buffer toeach well. The solution was moved carefully by pipetting down the sidesof the wells to avoid detaching the cells. Blocking was for 90 minutesat room temp with moderate shaking on a rotator and the two primaryantibodies were added to a tube containing Odyssey Blocking Buffer. Theprimary antibody was incubated for 2 hours with gentle shaking at roomtemp (Phosho-ERK (Rabbit, 1:100 dilution; Cell Signaling Technology9101). Total ERK2 (Mouse; 1:75 dilution; Santa Cruz BiotechnologySC-1647)). The wells were washed three times with 1×PBS+0.1% Tween-20for 5 minutes at room temp with gentle shaking, using a generous amountof buffer. The fluorescently labeled secondary antibody was diluted inOdyssey Blocking Buffer (Goat anti-rabbit Alexa Fluor 680 (1:200dilution; Molecular Probes) Goat anti-mouse IRDye 800CW (1:800 dilution;Rockland Immunochemicals)). The antibody solutions were mixed well and50 μL of the secondary antibody solution was added to each well. Theantibody solution was incubated for 60 minutes with gentle shaking atroom temp. The plate was washed three times with 1×PBS+0.1% Tween-20 for5 minutes at room temp with gentle shaking, using a generous amount ofbuffer. After a final wash, wash solution was completely removed fromwells. The plates were scanned with the Odyssey Infrared Imaging System(700 nm detection for Alexa Fluor 680 antibody and 800 nm detection forIRDye 800CW antibody). Negative control transfected cells yield 100% erkactivation (meaning background levels of active erk). Transfecting cellswith some of our miRNAs alters the level of active erk.

TABLE 19 miRNAs That Activate ERK miR % Activation Std Dev mir-218312.96 22.91 mir-210 291.74 38.23 mir-217 273.49 26.84 mir-152 265.5435.82 mir-148 264.38 43.55 mir-223 264.15 39.72 mir-301 261.36 61.77mir-328 259.48 45.87 mir-206 255.51 55.53 mir-125a 252.46 27.34mmu-mir-329 243.38 5.43 mir-19a 241.52 31.33 mir-25 238.90 44.94mmu-mir-294 235.51 24.60 mir-212 231.36 23.61 mmu-mir-295 221.47 14.05mir-370 220.60 22.88 mir-216 219.17 25.98 mir-96 213.93 57.07 mir-339213.9 42.25 mir-134 211.15 12.84 mir-372 211.13 5.67 Positive 245.3610.76 Control

Example 18 Screen for miRNAs that Influence Apoptosis

Apoptosis is a natural cellular process that helps control cancer byinducing death in cells with oncogenic potential. Many oncogenesfunction by altering induction of apoptosis. To identify miRNAs thatparticipate in apoptosis, an apoptosis assay was used with the miRNAinhibitor library.

HeLa cells (8000 cells/well of 96 well plate) were transfected intriplicate with more than 150 synthetic miRNAs (described above) (3pmoles) using Ambion siPORT™ NeoFX™. The media was changed 24 hrs aftertransfection and cells were processed 72 hrs after transfection. Thecells were measured for apoptosis by measuring caspase 3 activity asfollows: 1) Cells were washed once with PBS and frozen at −80° C. 2)Cells were lysed by adding 40 μl of cold lysis buffer (50 mM HEPES pH7.2, 40 mM NaCl, 0.5% NP40, 0.5 mM EDTA) to the wells and incubated for20 min at 4° C. 3) Add 160 ul ICE buffer (50 mM HEPES pH 7.4, 0.1%CHAPS, 0.1 mM EDTA, 10% sucrose)+5 mM DTT containing 20 uM DEVDafcsubstrate. 4) Measure fluorescence increase in one hour at 400 ex, 505em.

Samples were also analyzed for cell number using a general esteraseassay to normalize the caspase 3 results. FDA substrate (0.4 mg/mlfluorescein diacetate (FDA) in acetonitrile) was diluted 1:19 intodilution buffer (40 mM Tris Cl pH 7.5, 20 mM NaCl, 0.5% NP-40, 0.02mg/ml final conc). 40 μl buffer (40 mM Tris Cl pH 7.5, 0.5% NP-40) wasadded to each sample well. Samples were incubated 10 min on ice. 160 ulof diluted FDA substrate was added to each well. Fluorescence wasmeasured for 30 min at 37 deg (ex=488, em=529). The slope offluorescence increase over time is a function of the cell number in theplate.

miRNAs that affect apoptosis are listed in the table below. These miRNAsapparently regulate pathways that lead to apoptosis. Mis-regulation ofthese miRNAs could induce cells to undergo apoptosis or might keep thecells from undergoing apoptosis. Introducing or inhibiting these miRNAsin cancer (or other disease) cells that have overcome apoptoticsignaling pathways or Parkinson's (or other disease) cells that haveprematurely induced apoptosis could be used to treat the diseases.

TABLE 20 miRNAs that Significantly Increase the Percentage of ApoptoticCells Relative change in apoptotic cells St Dev mir-338 773.46 69.82mir-27a 607.24 150.08 mir-128 594.42 260.06 mir-23a 473.44 208.82mir-324 442.99 101.03 mir-22 439.13 62.59 mir-181a 409.97 65.14mmu-mir-293 403.86 53.41 mmu-mir-412 402.27 42.04 mir-196 378.13 28.15mir-31 373.90 61.39 Let-7d 369.10 88.94 mir-23b 360.68 81.97 mu-miR-290354.90 46.63 mir-217 347.38 56.49 mir-199 345.75 67.55 mir-24 317.4362.85 mir-214 312.25 7.38 mir-198 303.24 44.25

TABLE 21 miRNAs that Significantly Decrease the Percentage of ApoptoticCells Relative change in apoptotic cells St Dev mir-105 39.97 8.91mir-34a 37.75 8.41 mir-96 31.89 13.40 mmu-mir-292 30.72 4.27 mir-12628.71 4.24 mir-137 12.69 11.80 mir-101 7.50 6.91

Example 19 Synthetic miRNA Library Screen for miRNAs that InfluencehTert Expression

Telomerase is a complex of proteins and RNA that maintains the ends ofchromosomes by appending telomeres. With rare exceptions, terminallydifferentiated cells lack active telomerase. One of the exceptions iscancer cells. More than 90% of human cancer samples have activetelomerase (reviewed in Dong et al. 2005). The hTert gene encodes thecatalytic domain of telomerase. The expression of hTert correlates withtelomerase activity in cells making it a good surrogate for telomeraseactivity. An RT-PCR based assay for monitoring hTert mRNA expression intelomerase negative cells has been developed and used to identify miRNAsthat participate in the regulation of telomerase. The miRNAs thatregulate telomerase activity represent intervention points for cancertherapeutics.

BJ cells are normal human foreskin fibroblasts that lack hTert mRNA andtelomerase activity. BJ cells were trypsinized and diluted to 13,000cells/ml in normal growth media. 0.3 μl of lipofectamine 2000 agent wasdiluted into 40 μl of OPTI-MEM and incubated for five minutes. Thediluted transfection reagent was added to the wells of 96-well platesthat contained 150 synthetic miRNAs (as described above) as well as twodifferent negative control synthetic miRNAs. Each well housed adifferent synthetic miRNA. The synthetic miRNAs and transfection agentwere incubated for 15 minutes at room temperature and then 200 μl (2,600cells) were added on top of the lipid/miRNA complex. Cells were placedin an incubator and RNA was isolated 72 hours later. RNA was isolatedfrom the cells in each well using RNAqueous™-MagMAX96 Total RNAIsolation kit (Cat#1830) standard protocol (lyse cells in wells).Reverse transcription was done using the RETROscript reaction by adding11 μl of total RNA (20-100 ng/μl) to 1 ul of random decamers andincubated in 70° C. water bath for 3 minutes then place on ice. Next, 8ul of the cocktail containing Nuc-free water 3.8 μl, 10× ReverseTranscription buffer 2.0 ul, 2.5 mM dNTPs 2.0 ul, RNase InhibitorProtein (40 U/ul), 0.1 ul MMLV-RT (100 U/μl), and incubated at 42° C.for 1 hour, then 92° C. for 10 minutes.

Real time PCR reactions were assembled to quantify hTert mRNA and 18SrRNA in each of the samples. Nuclease-free water, 10× Complete PCRbuffer/SYBR, 25 mM MgCl2, 2.5 mM dNTPs, 50×ROX, 18S- or hTert-specificprimers (for & rev mix 3 uM), cDNA from the various samples, and Supertaq polymerase into a PCR tube. The reaction was heated to 95° C. for 5minutes and then subjected to 40 cycles of 95° C. for 15 seconds, 60° C.for 30 seconds, 72° C. for 30 seconds. The amplification products weremonitored using the ABI 7600 (Applied Biosystems). BJ cells ordinarilyfail to yield amplification products with the hTert primers. ThosemiRNA-transfected samples that yielded a hTert PCR product were alsoanalyzed for 18S rRNA lavels to ensure that there were not significantlymore cells in the samples that might have contributed to the amount ofhTert in the samples.

The hTert mRNA was detected in duplicate transfections of each of themiRNAs listed below. These miRNAs presumably affect pathways thatregulate the expression of the hTert gene. Over-expression of any ofthese miRNAs might contribute to cancer by activating telomerase.Regulating the activities of these miRNAs in cancer cells could limittheir transformation and overcome oncogenesis.

TABLE 22 hTert Activators mmu- mir-295 mir-92 mir-337 mir-26a mir-224mir-21 mir-195 mir-16 mir-15a mir-128 mir-125b mir-125a mir-105

Example 20 Synthetic miRNA Library Screens for miRNAs that InfluenceCell Cycle

The adult human body consists of about 50-100 trillion cells. Each day,several billion of these cells divide in two to replace the billions ofcells that die and are removed. In the course of an average lifetime,this adds up to an astronomical number of cell divisions, most of whichgo perfectly well. Errors do occur, however, and if they are notcorrected they may lead to cancer. Cell growth and division are normallycontrolled by an intricate system of checks and balances. Butoccasionally a cell will start to proliferate wildly, dividing again andagain and defying all normal restraints on its growth. That is thebeginning of most common forms of cancer.

The inventors transfected 4,000 BJ cells/well in triplicate with 46synthetic miRNAs using Lipofectamine 2000 (Invitrogen) according to themanufacturer's instructions. The

-   -   let7a    -   let7a    -   mir1    -   mir1    -   mir105    -   mir125a    -   mir128    -   mir142    -   mir145    -   mir146    -   mir147    -   mir150    -   mir15a    -   mir16    -   mir186    -   mir187    -   mir188    -   mir191    -   mir195    -   mir20    -   mir206    -   mir21    -   mir211    -   mir223    -   mir224    -   mir26a    -   mir320    -   mir324-3p    -   mir325    -   mir335    -   mir337    -   mir338    -   mir345    -   mir371    -   mir373    -   mir92    -   mmu201    -   mmu207    -   mmu290    -   mmu291-3p    -   mmu294    -   mmu295    -   mmu297    -   mmu322    -   mmu376b    -   mmu409

24 hours post-transfection, half of the BJ cells from each well wereremoved to fresh medium. 72 hrs post-transfection, the cells were fixedwith 4% paraformaldehyde at a final concentration of 2%. The fixed cellswere stained with propidium iodide (TTP LabTech protocol) and assessedusing the TTP LabTech cell scanner. Propidium iodide stains DNA and therelative DNA content in a cell corresponds with its position in the cellcycle. The cell scanner measured propidium iodide staining in each celland assigned its position in the cell cycle. The percentage of cells ineach stage of cell cycle was calculated and compared to cellstransfected with negative control synthetic miRNAs. The relative changein cells in each stage was calculated for each miRNA that was used.Those synthetic miRNAs that induced a significant shift toward or awayfrom a specific stage of cell cycle are listed below. These representmiRNAs that regulate key points in the cell cycle and offer keyintervention points for cancer-related therapeutic development.

TABLE 23 miRNAs that significantly reduce the percentage of BJ cells inG1 phase of the cell cycle miRNA % Diff in Cells in G1 St. Dev. mir-2154.4 4.2 mir-20 63.6 9.3 mir-1 65.3 9.5 mir-206 66.8 9.0 mir-373 72.65.7 mir-26a 78.0 4.0

TABLE 24 miRNAs that significantly increase the percentage of BJ cellsin G1 phase of the cell cycle miRNA % Diff in Cells in G1 St. Dev.mo-mir-325 121.7 5.3 mmu-409 123.2 13.7 mir-324 123.7 4.9 mir-195 125.12.5 mmu-376b 126.5 3.1 mir-142 127.0 13.0 mir-371 128.9 2.8 let-7a 131.54.5 mir-146 141.5 7.7 mir-128 143.0 2.4

TABLE 25 miRNAs that significantly reduce the percentage of BJ cells inS phase of the cell cycle miRNA % Diff in Cells in S St. Dev. mir-12855.5 3.8 let-7a 57.6 8.7 mir-142 59.5 24.7 mir-146 63.5 16.8 mmu-29765.0 14.1 mir-337 65.3 11.3 mir-195 65.6 0.1 mmu-376b 69.1 11.6 mir-32472.2 9.4 mir-187 72.3 10.9 mir-186 72.8 6.1

TABLE 26 miRNAs that significantly increase the percentage of BJ cellsin S phase of the cell cycle miRNA % Diff in Cells in S St. Dev. mir-92132.0 14.7 mir-15a 134.8 13.9 mir-191 135.9 29.1 mir-26a 136.0 7.6mir-20 139.7 17.6 mmu-290 141.0 11.7 let-7a 141.1 19.9 mir-345 143.345.8 mir-16 150.1 24.8 mir-224 150.6 9.8

TABLE 26 miRNAs that significantly reduce the percentage of BJ cells inG2/M phase of the cell cycle miRNA % Diff in Cells in G2/M St. Dev.mir-147 51.2 6.1 mir-371 52.8 2.7 mir-146 57.2 5.3 mir-195 58.9 4.4mir-128 65.4 2.7 mir-15a 67.4 13.7 let-7a 69.1 2.8

TABLE 27 miRNAs that significantly increase the percentage of BJ cellsin G2/M phase of the cell cycle miRNA % Diff in Cells in G2/M St. Dev.mir-26a 130.2 5.8 mir-187 132.0 4.3 mir-145 136.8 13.7 mir-373 137.9 5.2mir-20 143.0 10.6 mir-21 160.3 7.1

TABLE 28 miRNAs that significantly increase the percentage of BJ cellswith greater than 2 × amount of DNA miRNA % Diff in Cells w/>2 × DNA St.Dev. mir-20 157.9 23.4 mir-1 161.9 13.6 mir-345 176.1 17.4 mir-373 177.932.7 mir-337 195.0 52.1 mir-21 209.4 45.7

Example 21 Synthetic miRNA Library Screen for miRNAs that Influence CellProliferation

Cell proliferation assays were used in conjunction with our syntheticmiRNA library to identify miRNAs that influence cell proliferation in abroad range of cells, including those from lung, breast, prostate, skin,cervix, T-cell, and foreskin tissues.

Cervical (HeLa), lung (A549, CRL-5826, and HTB-57), breast (MCF12A andBT549), prostate (22Rv1), T-cells (Jurkat and primary normal), and skin(TE354T, TE353SK, and BJ) cells were transfected in triplicate with eachof the more than 150 synthetic miRNAs in our library. With theexceptions of Jurkats and Primary T-cells, each cell type wastransfected with 5 picomoles of each of the miRNAs in the syntheticmiRNA library using siPORT™ NeoFX™ (Ambion) at a plating density ofapproximately 8000 cells/well of 96 well plate. The Jurkats and primaryT-cells were mixed at a rate of approximately 50,000 cells/well with 500picomoles of each of the synthetic miRNAs. The media was changed 24 hrsafter transfection. 72 hours post-transfection, cell number wasestimated by one of three methods:

-   -   (1) Alamar blue was added to each well and the 96-well plates        were analyzed using a plate reader. Alamar blue is a substrate        for a metabolic enzyme in cells and the reaction product is        fluorescent. The fluorescence in each well correlates with the        total number of cells in each well.    -   (2) ViaCount Flex Reagent (Guava), a dye that fluoresces when it        interacts with DNA, was added to each well and fluorescence was        quantified using the Guava PCA-96 according to the        manufacturer's instructions.    -   (3) Propidium iodide, a dye that fluoresces when it interacts        with DNA, was added to each well and the total number of cells        in the well was estimated by counting unique sites of stained        DNA using the TTP LabTech Cell Scanner according to the        manufacturer's instructions.        The impact of each miRNA on cell proliferation was assessed by        dividing the cell number reading of each well by the average        cell number reading for wells transfected with a negative        control (NC) miRNA.

Presented in FIG. 15A-C are synthetic miRNAs that significantly reducedthe proliferation of the various cell types that were analyzed. ThesemiRNAs represent molecules that could be used for therapeutics,diagnostics, creating cell lines with interesting research properties,and inducing differentiation.

Approximately 10% of the miRNAs significantly reduced cell proliferationfor at least four different cell types. These miRNAs (presented inranked order in the table below) are provided below and can beimplemented in methods and compositions of the invention.

TABLE 29 Common Anti-Proliferation miRNAs miRNA # Positives miR-124 7miR-16 6 miR-101 6 miR-126 6 miR-147 6 miR-15a 5 miR-96 5 miR-105 5miR-142 5 miR-215 5 miR-346 4 miR-206 4 miR-192 4 miR-194 4

Among the cells that were used in the synthetic miRNA library screensare matched pairs of cancer and non-cancer cells from breast, skin, andT-cell. Interestingly, many synthetic miRNAs differentially affectedproliferation in the cell pairs (see table below).

TABLE 30 Breast Non- Cancer Cancer miRNA % NC % Std Dev % NC % Std Devmir-201 79 14 103 17 mir-192 81 3 95 17 mir-92 85 11 104 24 Skin CancerNormal pre-MIR % of NC % ST DEV % of NC % ST DEV mir-154 51 5 93 10mir-195 58 3 87 5 mu-mir-376b 65 3 99 8 mir-201 67 8 106 4 mir-26a 69 1297 17 mir-193 69 4 105 10 T-Cell Leukemia Normal % NC % St Dev % NC % StDev let-7a 21 1 137 15 let-7b 50 5 136 13 miR-101 69 30 95 5 miR-10b 373 115 18 miR-122 67 18 104 18 miR-17-3p 63 16 116 4 miR-29a 68 7 111 8miR-30a-3p 66 27 97 18 miR-34a 67 21 100 1Presented in FIG. 16 are synthetic miRNAs that significantly increasethe proliferation of the various cell types that were analyzed.

Example 22 miRNA Inhibitor Library Screens Identify miRNAs thatInfluence Cell Proliferation

A cell proliferation assay was used in conjunction with our syntheticmiRNA library to identify miRNAs that influence cell proliferation in abroad range of cells, including those from lung, breast, prostate, skin,cervix, T-cell, and foreskin tissues.

Breast (MCF12A), prostate (22Rv1), lung (A549), and skin (TE354T) cellswere transfected in triplicate with each of the more than 150 miRNAinhibitors in our library. Each cell type was transfected with 10picomoles of each of the miRNA inhibitors in the library using siPORT™NeoFX™ (Ambion) at a plating density of approximately 8000 cells/well of96 well plate. 72 hours post-transfection, cell number was estimated byone of three methods:

-   -   (1) Alamar blue was added to each well and the 96-well plates        were analyzed using a plate reader. Alamar blue is a substrate        for a metabolic enzyme in cells and the reaction product is        fluorescent. The fluorescence in each well correlates with the        total number of cells in each well.    -   (2) ViaCount Flex Reagent (Guava), a dye that fluoresces when it        interacts with DNA, was added to each well and fluorescence was        quantified using the Guava PCA-96 according to the        manufacturer's instructions.    -   (3) Propidium iodide, a dye that fluoresces when it interacts        with DNA, was added to each well and the total number of cells        in the well was estimated by counting unique sites of stained        DNA using the TTP LabTech Cell Scanner according to the        manufacturer's instructions.

The impact of each miRNA inhibitor on cell proliferation was assessed bydividing the cell number reading of each well by the average cell numberreading for wells transfected with a negative control (NC) miRNA.

Presented in FIG. 17 are miRNAs whose inhibition significantly reducedthe proliferation of the various cell types that were analyzed. ThesemiRNAs represent molecules that could be used for therapeutics,diagnostics, creating cell lines with interesting research properties,and inducing differentiation.

Presented in FIG. 18 are miRNA inhibitors that significantly increasethe proliferation of the various cell types that were analyzed. ThesemiRNAs represent molecules that could be used for therapeutics,diagnostics, creating cell lines with interesting research properties,and inducing differentiation.

Example 23 Synthetic miRNA Library Screen for miRNAs that Influence CellViability

The basis for most human diseases is the subversion of one or more cellsto function in ways that are outside what they normally do. Forinstance, cancer initiates with the immortalization and transformationof a single cell which then divides repeatedly to form a tumor.Compounds that reduce the viability of disease cells are used routinelyto treat patients with cancer and other diseases.

Cervical (HeLa), lung (A549), and T-cells (Jurkat and primary normal)were transfected in triplicate with each of the more than 150 syntheticmiRNAs in our library. With the exceptions of Jurkats and PrimaryT-cells, each cell type was transfected with 5 picomoles of each of themiRNAs in the synthetic miRNA library using siPORT™ NeoFX™ (Ambion) at aplating density of approximately 8000 cells/well of 96 well plate. TheJurkats and primary T-cells were mixed at a rate of approximately 50,000cells/well with 500 picomoles of each of the synthetic miRNAs. For theHeLa and A549 cells, the media was changed 24 hrs after transfection. 72hours post-transfection, cell viability was estimated by one of twomethods:

-   -   (1) ViaCount Flex Reagent (Guava), which includes a dye that can        only enter dead cells and that fluoresces when it interacts with        DNA, was added to each well and fluorescence was quantified        using the Guava PCA-96 according to the manufacturer's        instructions. The percentage of viable cells was measured by        dividing the number of non-dead and non-apoptotic cells in the        sample by the total number of cells in the well and multiplying        by 100.    -   (2) Propidium iodide, a dye that fluoresces when it interacts        with DNA, was added to each well. Each cell was analyzed using        the TTP LabTech Cell Scanner according to the manufacturer's        instructions to detect cells with staining patterns consistent        with cell death or apoptosis. The percentage of viable cells was        measured by dividing the number of non-dead and non-apoptotic        cells in the sample by the total number of cells in the well and        multiplying by 100.

Presented in FIG. 19 are synthetic miRNAs that significantly decrease orincrease viability in the various cell types that were analyzed. Acomparison of the viability of jurkat and primary T-cells, whichrepresent the leukemic and normal forms of T-cells, let-7, miR-10,miR-101, miR-17-3p, miR-19, and miR-34a severely reduced the viabilityof the leukemia cells without adversely affecting the normal T-cells.

Example 24 Synthetic miRNA Library Screen for miRNAs that InfluenceApoptosis

To identify miRNAs that participate in apoptosis, an apoptosis assay wasused with the miRNA inhibitor library.

Approximately 8000 cervical (HeLa), prostate (22Rv1), T-cell (Jurkat),and skin (TE354T) cells per well were transfected in triplicate witheach of the more than 150 synthetic miRNAs in our library using siPORT™NeoFX™ (Ambion). Media was changed after 24 hrs and cells were visuallyinspected under a microscope to qualitatively inspect cell death 72hours after transfection. The cells were measured for apoptosis bymeasuring caspase 3 activity as follows: 1) Cells were washed once withPBS and frozen at −80° C. 2) Cells were lysed by adding 40 μl of coldlysis buffer (50 mM HEPES pH 7.2, 40 mM NaCl, 0.5% NP40, 0.5 mM EDTA) tothe wells and incubated for 20 min at 4° C. 3) Add 160 μl CE buffer (50mM HEPES pH 7.4, 0.1% CHAPS, 0.1 mM EDTA, 10% sucrose)+5 mM DTTcontaining 20 μM DEVDafc substrate. 4) Measure fluorescence increase inone hour at 400 ex, 505 em. Samples were also analyzed for cell numberusing a general esterase assay to normalize the caspase 3 results. FDAsubstrate (0.4 mg/ml fluorescein diacetate (FDA) in acetonitrile) wasdiluted 1:19 into dilution buffer (40 mM Tris CI pH 7.5, 20 mM NaCl,0.5% NP-40, 0.02 mg/ml final conc). 40 μl buffer (40 mM Tris Cl pH 7.5,0.5% NP-40) was added to each sample well. Samples were incubated 10 minon ice. 160 μl of diluted FDA substrate was added to each well.Fluorescence was measured for 30 min at 37 deg (ex=488, em=529). Theslope of fluorescence increase over time is a function of the cellnumber in the plate.

The impact of each miRNA on apoptosis was assessed by dividing thecaspase 3 reading of each well by the average caspase 3 reading forwells transfected with a negative control (NC) miRNA.

As seen in FIG. 20, many different miRNAs were able to increase ordecrease apoptosis in the four cell types that were analyzed. A fewmiRNAs (miR-126, miR-26a, miR-1, miR-149, and let-7g) affected apoptosisin multiple cell types suggesting that they regulate apoptosis via genesthat are common in multiple cell types.

Example 25 Synthetic miRNA Library Screen for miRNAs that InduceTransformation

Transformation is necessary for tumor formation as it overcomes thecell's natural response to stop dividing when placed in a crowdedenvironment. To identify miRNAs that participate in transformation, atransformation assay featuring NIH3T3 cells was used with the syntheticmiRNA library. NIH3T3 cells are used in transformation assays as theylack the capacity to form colonies when plated in soft agar. Modulationof cell processes that inhibit transformation can be readily detectedbecause they induce NIH3T3 cells to begin forming colonies when platedin soft agar.

Approximately 8000 NIH 3T3 cells were transfected in duplicate with eachof the more than 150 synthetic miRNAs in our library using siPORT™NeoFX™ (Ambion). Media was changed after 24 hrs and the cells weretransferred to 24-well dishes containing soft agar. The soft agar limitsmobility and ensures that sister cells must remain in contact followingcell division. Close contact with other cells typically induces the NIH3T3 cells to stop dividing. The total number of cells in each well wasmeasured by taking an absorbance reading at 495 nm. The absorbancereading for each well was divided by the average absorbance reading forcells transfected with negative control miRNAs and multiplied by 100 toget the percent change in transformation. An initial screen revealedmiR-10, miR-23, miR-24, miR-198, miR-192, and miR-199 as miRNAs thatincreased transformation relative to cells transfected with negativecontrol. A repeat of the experiment with the initial candidates yieldedthe following hit as shown below:

TABLE 31 miRNA % NC % SD 198 103 2.07 192 108 5.7 199 113 5.59

Example 26

miRNAs that Affect the Efficacy of Therapeutic Compounds

Many compounds have been tested in clinical trials for their capacity topositively affect the outcome of patients. In some cases, thesecompounds meet the standards set for by the FDA and they becometherapeutics. Unfortunately, very few therapeutics are 100% effective.Enhancing the activities of therapeutic compounds provides a significantopportunity within the medical industry. The two most common methodsthat are used to enhance therapeutics are modifying the chemicalstructure of the compounds or using multiple therapeutic compoundssimultaneously. Whether it would be beneficial to introduce miRNAs inadvance of adding compounds that are known to significantly reduce theviability of cancer cells was evaluated. One of the anti-cancercompounds that was introduced was TRAIL, a compound that binds at leasttwo different receptors and activates the apoptosis pathway to inducecell death primarily in cancer cells. The second compound that wastested in combination with synthetic miRNAs was etoposide, atopoisomerase II inhibitor that activates the apoptosis pathway ofcancer and normal cells alike by reducing the repair of DNA damagewithin the cells.

Approximately 8000 cervical (HeLa) and lung (A549, HTB-57, and CRL-5826)cells per well were transfected in triplicate with synthetic miRNAs fromour library using siPORT™ NeoFX™ (Ambion). Media was changed after 24hrs and etoposide and TRAIL were introduced at a final concentration ofapproximately 25 μM after 48 hours. The cells were visually inspectedunder a microscope to qualitatively inspect cell death 64 hours aftertransfection.

The cells treated with etoposide were measured for apoptosis bymeasuring caspase 3 activity as follows: 1) Cells were washed once withPBS and frozen at −80° C. 2) Cells were lysed by adding 40 μl of coldlysis buffer (50 mM HEPES pH 7.2, 40 mM NaCl, 0.5% NP40, 0.5 mM EDTA) tothe wells and incubated for 20 min at 4° C. 3) Add 160 μl ICE buffer (50mM HEPES pH 7.4, 0.1% CHAPS, 0.1 mM EDTA, 10% sucrose)+5 mM DTTcontaining 20 μM DEVDafc substrate. 4) Measure fluorescence increase inone hour at 400 ex, 505 em. Samples were also analyzed for cell numberusing a general esterase assay to normalize the caspase 3 results. FDAsubstrate (0.4 mg/ml fluorescein diacetate (FDA) in acetonitrile) wasdiluted 1:19 into dilution buffer (40 mM Tris Cl pH 7.5, 20 mM NaCl,0.5% NP-40, 0.02 mg/ml final conc). 40 μl buffer (40 mM Tris Cl pH 7.5,0.5% NP-40) was added to each sample well. Samples were incubated 10 minon ice. 160 μl of diluted FDA substrate was added to each well.Fluorescence was measured for 30 min at 37 deg (ex=488, em=529). Theslope of fluorescence increase over time is a function of the cellnumber in the plate.

The cells treated with TRAIL were assessed for cell viability by addingalamar blue each well and analyzing fluorescence using a plate reader.Alamar blue is a substrate for a metabolic enzyme in cells and thereaction product is fluorescent. The fluorescence in each wellcorrelates with the total number of cells in each well.

The effect of each miRNA on the treatments was measured by dividing thecaspase 3 or alamar blue reading of the cells transfected with miRNAsand treated with TRAIL or etoposide by the same readings for cells thatwere only transfected with the miRNAs. The change in caspase 3 activityor alamar blue staining for each miRNA was then divided by thedifferences observed for two negative control miRNAs and multiplied by100 to calculate the relative effect induced by the combination of eachmiRNA and the therapeutic compound. These values are listed as % NC inFigure G.

As shown in FIG. 21, a number of miRNAs significantly increased thecapacity of the two therapeutic compounds to induce cell death in thecancer cells that were treated. Interestingly, mir-292-3p, mir-132,mir-124, and mir-28 all worked extremely well in combination with bothTRAIL and etoposide.

Example 27 Synthetic miRNA Library Screen for miRNAs that Affect CellCycle

The adult human body consists of about 50-100 trillion cells. Each day,several billion of these cells divide in two to replace the billions ofcells that die and are removed. In the course of an average lifetime,this adds up to an astronomical number of cell divisions, most of whichgo perfectly well. Errors do occur, however, and if they are notcorrected they may lead to cancer. Cell growth and division are normallycontrolled by an intricate system of checks and balances. Butoccasionally a cell will start to proliferate wildly, dividing again andagain and defying all normal restraints on its growth. That is thebeginning of most common forms of cancer.

Approximately 8000 cervical (HeLa) and 4000 skin (BJ) cells per wellwere transfected in triplicate with each of the more than 150 syntheticmiRNAs in our library. HeLa cells were transfected using siPORT™ NeoFX™(Ambion) and BJ cells were transfected using Lipofectamine 2000(Invitrogen) according to the manufacturer's instructions. 24 hourspost-transfection, half of the cells from each well were removed tofresh medium. 72 hrs post-transfection, the cells were fixed with 4%paraformaldehyde at a final concentration of 2%. The fixed cells werestained with propidium iodide (TTP LabTech protocol) and assessed usingthe TTP LabTech cell scanner. Propidium iodide stains DNA and therelative DNA content in a cell corresponds with its position in the cellcycle. The cell scanner measured propidium iodide staining in each celland assigned its position in the cell cycle. The percentage of cells ineach stage of cell cycle was calculated and compared to cellstransfected with negative control synthetic miRNAs. The relative changein cells in each stage was calculated for each miRNA that was used.Those synthetic miRNAs that induced a significant shift toward or awayfrom a specific stage of cell cycle are listed below. These representmiRNAs that regulate key points in the cell cycle and offer keyintervention points for cancer-related therapeutic development.

As seen in FIG. 22, many different miRNAs significantly altered thepercentage of cells in the various stages of cell cycle in the two celltypes that were analyzed.

Example 28 Synthetic miRNA Library Screen for miRNAs that Influence ERKActivity

Inventors: is this the Same as Example 17?

In order for cancer cells to proliferate they must subvert both themachinery that controls the cell division cycle and the process ofprogrammed cell death (apoptosis). This is frequently achieved bymutation of specific proto-oncogenes such as Ras or tumor suppressorssuch as p53. The Ras-family of membrane associated GTPases transmitsignals into the interior of the cell by the activation of a number ofcytosolic signal transduction pathways such as the Raf>MEK>ERK MAPkinase signaling pathway. Disregulation of the Ras/Raf/MEK/ERK pathwayplays a major role in cancer pathogenesis (reviewed in Meijer et al.).

To identify miRNAs that affect ERK activation, HeLa cells weretransfected in a 96-well plate format with 160 different syntheticmiRNAs. Prior to transfection, the HeLa cells were trypsinized to removeadherent cells and diluted in normal growth medium to 10⁵ cells/mL. 0.5μl of siPort NeoFX in 9.5 μl of Optimem I medium was added to the cellsand incubated for 10 minutes at room temp (10 μL for each sample).miRNAs were rehydrated with 10 μl of diluted siPORT NeoFX. The sampleswere incubated at 37° C. and then the transfected samples were evaluated72 hours after transfection.

The controls for ERK activation were performed by depriving the wells ofa phosphate source for detection of ERK phosphorylation. 100 μl ofserum-free media (DMEM) to 37° C. was added per well and the cells wereincubated for 4 hours at 37° C. to attain basal phosphorylation levels.For the positive control wells, serum-free media was aspirated fromwells and 100 μL of 100 ng/mL EGF was added before incubating the cellsfor 7.5 minutes at 37° C.

Media from all wells was removed by aspiration and the cells wereimmediately fixed in 150 μL of 3.7% Formaldehyde in 1×PBS for 20 minutesat room temp with no shaking. Fixing solution was removed to anappropriate waste container. The fixed cells were washed three timeswith 1×PBS. The wells were then washed three times with 200 μL of 1×PBScontaining 0.1% Triton X-100 for 5 minutes per wash, with shaking atroom temp.

Cells were blocked by adding 150 μL of Li—COR Odyssey Blocking Buffer toeach well. The solution was moved carefully by pipetting down the sidesof the wells to avoid detaching the cells. Blocking was for 90 minutesat room temp with moderate shaking on a rotator and the two primaryantibodies were added to a tube containing Odyssey Blocking Buffer. Theprimary antibody was incubated for 2 hours with gentle shaking at roomtemp (Phosho-ERK (Rabbit, 1:100 dilution; Cell Signaling Technology9101). Total ERK2 (Mouse; 1:75 dilution; Santa Cruz BiotechnologySC-1647)). The wells were washed three times with 1×PBS+0.1% Tween-20for 5 minutes at room temp with gentle shaking, using a generous amountof buffer. The fluorescently labeled secondary antibody was diluted inOdyssey Blocking Buffer (Goat anti-rabbit Alexa Fluor 680 (1:200dilution; Molecular Probes) Goat anti-mouse IRDye 800CW (1:800 dilution;Rockland Immunochemicals)). The antibody solutions were mixed well and50 μL of the secondary antibody solution was added to each well. Theantibody solution was incubated for 60 minutes with gentle shaking atroom temp. The plate was washed three times with 1×PBS+0.1% Tween-20 for5 minutes at room temp with gentle shaking, using a generous amount ofbuffer. After a final wash, wash solution was completely removed fromwells. The plates were scanned with the Odyssey Infrared Imaging System(700 nm detection for Alexa Fluor 680 antibody and 800 nm detection forIRDye 800CW antibody).

TABLE 32 miRNAs That Activate ERK miRNA % NC % StDev let-7 250 25mir-125a 252 27 mir-134 211 13 mir-148 264 44 mir-152 266 36 mir-19a 24231 mir-206 256 56 mir-207 224 3 mir-210 292 38 mir-212 231 24 mir-216219 26 mir-217 273 27 mir-218 313 23 mir-223 264 40 mir-25 239 45mir-294 236 25 mir-295 221 14 mir-301 261 62 mir-328 259 46 mir-329 2435 mir-339 214 42 mir-370 221 23 mir-372 211 6 mir-96 214 57 PositiveControl 245 11

Example 29 Synthetic miRNA Library Screen for miRNAs that InfluencehTert Expression

Telomerase is a complex of proteins and RNA that maintains the ends ofchromosomes by appending telomeres. With rare exceptions, terminallydifferentiated cells lack active telomerase. One of the exceptions iscancer cells. More than 90% of human cancer samples have activetelomerase (reviewed in Dong et al., 2005). The hTert gene encodes thecatalytic domain of telomerase. The expression of hTert correlates withtelomerase activity in cells making it a good surrogate for telomeraseactivity. We have developed and used an RT-PCR based assay formonitoring hTert mRNA expression in telomerase negative cells toidentify miRNAs that participate in the regulation of telomerase. ThemiRNAs that regulate telomerase activity represent intervention pointsfor cancer therapeutics.

BJ cells are normal foreskin fibroblasts that lack hTert mRNA andtelomerase activity. BJ cells were trypsinized and diluted to 13,000cells/ml in normal growth media. 0.3 μl of lipofectamine 2000 agent wasdiluted into 40 μl of OPTI-MEM and incubated for five minutes. Thediluted transfection reagent was added to the wells of 96-well platesthat contained 151 synthetic miRNAs as well as two different negativecontrol synthetic miRNAs. Each well housed a different synthetic miRNA.The synthetic miRNAs and transfection agent were incubated for 15minutes at room temperature and then 200 μl (2,600 cells) were added ontop of the lipid/miRNA complex. Cells were placed in an incubator andRNA was isolated 72 hours later. RNA was isolated from the cells in eachwell using RNAqueous™-MagMAX96 Total RNA Isolation kit (Cat#1830)standard protocol (lyse cells in wells). Reverse transcription was doneusing the RETROscript reaction by adding 11 ul of total RNA (20-100ng/μl) to 1 μl of random decamers and incubated in 70° C. water bath for3 minutes then place on ice. Next, 8 μl of the cocktail containingNuc-free water 3.8 μl, 10× Reverse Transcription buffer 2.0 μl, 2.5 mMdNTPs 2.0 μl, RNase Inhibitor Protein (40 U/μl), 0.1 μl MMLV-RT (100U/μl), and incubated at 42° C. for 1 hour, then 92° C. for 10 minutes.

Real time PCR reactions were assembled to quantify hTert mRNA and 18SrRNA in each of the samples. Nuclease-free water, 10× Complete PCRbuffer/SYBR, 25 mM MgCl2, 2.5 mM dNTPs, 50×ROX, 18S- or hTert-specificprimers (for & rev mix 3 μM), cDNA from the various samples, and Supertaq polymerase were placed into a PCR tube. The reaction was heated to95° C. for 5 minutes and then subjected to 40 cycles of 95° C. for 15seconds, 60° C. for 30 seconds, 72° C. for 30 seconds. The amplificationproducts were monitored using the ABI 7600 (Applied Biosystems). BJcells ordinarily fail to yield amplification products with the hTertprimers. Those miRNA-transfected samples that yielded a hTert PCRproduct were also analyzed for 18S rRNA lavels to ensure that there werenot significantly more cells in the samples that might have contributedto the amount of hTert in the samples.

The hTert mRNA was detected in duplicate transfections of each of themiRNAs listed below. These miRNAs presumably affect pathways thatregulate the expression of the hTert gene. Over-expression of any ofthese miRNAs might contribute to cancer by activating telomerase.Regulating the activities of these miRNAs in cancer cells could limittheir transformation and overcome oncogenesis.

TABLE 33 hTert miRNA Activators Log(2) hTert miRNA Expression mir-1473.14 mir-195 4.25 miR-21 1.55 mir-24 4.68 mir-26a 4.35 mir-301 4.14mir-368 5.30 mir-371 2.43

The telomerase activity screen was repeated using a series of siRNAstargeting kinases, phosphatases, GPCRs, transcription factors, andassorted other genes. Targeting the genes below with siRNAs resulted inincreased hTert expression. Interestingly, many of these genes arepredicted to be targets for the miRNAs that we found to be hTertregulators (see table below).

TABLE 34 hTert Gene Activators Log(2) hTert Gene Expression ACOX1 3.44AKT1 1.80 APAF1 3.40 COX-5B 2.78 COX6 2.28 COX7B 3.95 CPOX 4.66 DUOX23.80 GPX1 1.85 GPX2 2.56 GPX4 3.17 LPO 3.37 MAPK1 3.07 MAPK4 3.61 MTCO11.58 NOX3 2.30 NOX5 2.54 PAOX 1.72 PPOX 2.09 PRKCA 2.24 PRKCD 4.39TNFRSF6 2.25

Example 30 Effect of miRNA Primary Sequence on Function

Many miRNAs appear to be very closely related to others based on theirprimary sequences. For instance, let-7a is a member of the let-7 genefamily, which includes 7 unique genes within the human genome. The let-7genes encode miRNAs that vary by as little as a single nucleotide and asmany as four nucleotides. In our synthetic miRNA and miRNA inhibitorlibraries, we have five different human let-7 miRNAs. These miRNAs havebeen used in many different cell types in screens designed to identifymiRNAs involved in a variety of different cellular processes. In many ofthe screens, the various let-7 miRNAs generate similar phenotypes. FIG.223 provides two examples wherein all of the let-7 family members yieldsimilar responses. In contrast, there are some screens wherein thevarious let-7 family miRNAs yield significantly different results (FIG.23).

Example 31 Synthetic miRNA Library Screen for miRNAs that InfluenceInflammation

Inflammation is the body's natural protective response to an injury orinfection. It is designed to hyper-stimulate biological pathways thatinitiate tissue repair or attack invading pathogens. This response is adelicate balance of both pro- and anti-inflammatory genes and theirproteins. If the inflammatory response is maintained too long it canlead to tissue destruction, organ failure or inflammatory diseases suchas Rheumatoid arthritis, Psoriasis, Asthma, Inflammatory bowel disease(Crohn's disease and related conditions), Multiple Sclerosis, coronaryobstructive pulmonary disease (COPD), Allergic rhinitis (hay fever), andCardiovascular disease.

Stat3 is the subject of intense scientific investigation, because it'sknown to be an important transcription factor that turns on genesrequired for the cell division, induction and suppression of apoptosis,and cell motility. Many STAT3 target genes are known, including thoseencoding the anti-apoptotic proteins Bcl-x1, Mcl-1, and Bcl-2, theproliferation-associated proteins Cyclin D1 and Myc, and thepro-angiogenic factor VEGF. The inflammatory disease psoriasis ischaracterized by lesions, which contain epidermal keratinocytes thatexpress high levels of activated Stat3. Stat3 has also recently beendiscovered to play an important role as an anti-inflammatory regulator.In normal mice, the immune system is initially upregulated in responseto bacterial protein challenge creating systemic inflammation followedby down regulation of the initiating factors. Mice with a deletionalmutation for Stat3-beta lacked the ability to down regulate the initialinflammatory reaction after bacterial protein challenge which lead toirreversible damage to the animals' own tissues and finally to animaldeath.

A stat3 response assay was used to identify miRNAs that regulatecellular inflammatory response. The stable Stat3-luciferase reportercell line from Panomics, which contains a chromosomal integration of aluciferase reporter construct regulated by 3 copies of the Stat1response element was used for this purpose. The chemical agentPhorbol-12-myristate 13 acetate (PMA) is known to induce an inflammatoryresponse in exposed cells and was used to stimulate inflammation in thisexperiment. These cells were transfected in triplicate with each of themore than 206 synthetic miRNAs in our library using siPORT™ NeoFX™(Ambion) at a plating density of approximately 6000 cells/well of 96well plate. The media was changed 24 h post transfection and exposed to100 nM PMA for 6 hours starting at 67 hours post transfection. The cellswere assayed for changes in total cell number by alamarBlue aspreviously described and finally harvested at 72 hours post initialtransfection. A luciferase assay was performed on all sample lysates tomeasure Stat3 responsiveness to the procedure. The data was normalizedto total cell number using the alamar Blue data and compared to cellstransfected with a negative control miRNA that underwent the sameprocedure.

The following miRNA were able to reduce the ability of PMA to stimulateStat3.

% of NC % STDEV mir-93 34 74 mir-100 13 10 mir-134 50 18 mir-99a 38 96mir-103 38 40 mir-128 49 115 mir-129 44 112 mir-181b 11 21 mir-193 42 92mir-197 36 78 mir-212 42 92 mir-218 38 84 mir-219 39 86 mir-302 40 87mir-323 22 49 mir-324-3p 29 63 mir-325 29 63 mir-330 21 47 mir-331 39 86mir-340 34 75 mmu-mir-350 11 22 mir-425 24 49 mir-491 25 49 mir-518f 2652 mir-520a* 28 55

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of embodiments, it will be apparent to those ofskill in the art that variations may be applied to the compositions andmethods, and in the steps or in the sequence of steps of the methods,described herein without departing from the concept, spirit, and scopeof the invention. More specifically, it will be apparent that certainagents that are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references are specifically incorporated by reference tothe extent they relate to topics and subject matter discussed herein.

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1.-78. (canceled)
 79. A method for treating cancer in a subjectcomprising administering to the subject an effective amount of one ormore synthetic miRNA molecules and/or miRNA inhibitors corresponding tomiR-21, miR-126, miR-143, miR-145, miR-188, miR-200b, miR-219, ormiR-331.
 80. The method of claim 79, wherein the cancer is breast cancerand the subject is administered 1) one or more miRNA inhibitorscorresponding to miR-21, miR-15a, miR-16, miR-24, and/or miR-25, and/or2) one or more miRNAs corresponding to miR-99, miR-100, miR-205,miR-197, miR-126, miR-143, miR-145 and/or miR-321.
 81. The method ofclaim 79, wherein the cancer is colon cancer and the subject isadministered 1) one or more miRNA inhibitors corresponding to miR-21,miR-106, miR-189, miR-200b, miR-223, miR-224, miR-31, and/or miR-17;and/or 2) one or more miRNAs corresponding to miR-145, miR-143, miR-133,miR-342, miR-125a, miR-195, miR-30a, miR-10a, miR-130, miR-130a,miR-192, miR-194, miR-215, miR-144, miR-23, miR-26a, miR-126, miR-199a,miR-188, miR-331, and/or miR-21.
 82. The method of claim 79, wherein thecancer is thyroid cancer and the subject is administered 1) one or moremiRNA inhibitors corresponding to miR-21 miR-125, miR-24, miR-200b,miR-29b, miR-221, miR-222, miR-224, miR-10a, and/or miR-183; and/or 2)one or more miRNAs corresponding to miR-145, miR-22, miR-331, miR-126,miR-30a, miR-199a, miR-223, and/or miR-321.
 83. The method of claim 79,wherein the cancer is lung cancer and the subject is administered 1) oneor more miRNA inhibitors corresponding to miR-223, miR-106, miR-21,miR-200b, miR-321, miR-182, miR-183, miR-17, and/or miR-205; and/or 2)one or more miRNAs corresponding to miR-130a, miR-145, miR-126, miR-331,miR-342, miR-143, Let-7, miR-30a, miR-16, miR-26a, miR-125a, miR-29b,miR-24, miR-328, miR-201, miR-195, miR-22, miR-181a, miR-331, and/ormiR-321.
 84. A method for treating cancer in a subject comprisingadministering to the subject an effective amount of one or moresynthetic miRNA molecules and/or miRNA inhibitors corresponding tomiR-10a, miR-16, miR-17, miR-21, miR-22, miR-23, miR-24, miR-26a,miR-29b, miR-30a, miR-106, miR-125a, miR-126, miR-130, miR-130a,miR-133, miR-143, miR-144, miR-145, miR-181a, miR-182, miR-183, miR-188,miR-189, miR-219, miR-192, miR-194, miR-195, miR-199a, miR-200b,miR-201, miR-205, miR-215, miR-223, miR-224, miR-321, miR-328, miR-331,or miR-342.
 85. A method of treating cancer in a patient comprisingadministering to the patient the cancer therapeutic and an effectiveamount of at least one miRNA molecule that improves the efficacy of thecancer therapeutic or protects non-cancer cells.
 86. The method of claim85, wherein the miRNA molecule enhances the efficacy of the cancertherapeutic and is selected from the group consisting of ambi-miR-7100,mir-28, mir-101, mir-124, mir-125a, mir-126, mir-132, mir-136, mir-147,mir-155, mir-182, mir-186, mir-202, mir-206, mir-216, mir-221, mir-224,mir-291, mir-292-3p, mir-297, mir-302, mir-337, mir-372, mir-373, andmir-376b.
 87. The method of claim 85, wherein the miRNA moleculeprotects non-cancer cells from the cancer therapeutic and is selectedfrom the group consisting of mir-16, mir-24, mir-30a-3p, mir-125b,mir-152, mir-194, mir-197, mir-214, and mir-331. 88.-110. (canceled)102. The method of claim 79, wherein the cancer is a malignant cancer, atumor, a metastatic cancer, an unresectable cancer, a chemo- and/orradiation-resistant cancer, or a terminal cancer.